Stable Liquid Antibody Formulation

ABSTRACT

The present invention relates generally to the field of pharmaceutical formulations of antibodies. Specifically, the present invention relates to a stable liquid antibody formulation and its pharmaceutical preparation and use. This invention is exemplified by a liquid formulation of a humanised anti-NGF antibody.

FIELD OF THE INVENTION

The present invention relates to the field of pharmaceuticalformulations of antibodies. Specifically, the present invention relatesto a stable liquid antibody formulation and its pharmaceuticalpreparation and use.

BACKGROUND OF THE INVENTION

Antibody preparations intended for therapeutic or prophylactic userequire stabilizers to prevent loss of activity or structural integrityof the protein due to the effects of denaturation, oxidation oraggregation over a period of time during storage and transportationprior to use. These problems are exacerbated at the high concentrationsof antibody often desired for therapeutic administration.

A major aim in the development of antibody formulations is to maintainantibody, solubility, stability and potency of its antigen binding. Itis particularly desirable to avoid aggregates and particulates insolution which would require sterile filtration before use forintravenous or subcutaneous injection and limit route of administration.Antibody aggregates can cause pain and anaphylactoid side effects whenthe formulation containing them is intravenously injected.

Lyophilisation and freeze drying are alternatives to the liquidformulation of antibodies. Both processes have a propensity for inducingdenaturation of the antibody and decreasing of its antigen-bindingactivity particularly upon reconstitution.

Salts, surfactants, pH and tonicity agents such as sugars can contributeto overcoming aggregation problems. Formulation of antibody preparationsrequires careful selection of these factors among others to avoiddenaturation of the protein and loss of antigen-binding activity.Regarding a pH range of an antibody preparation, if an antibodyformulation having a low pH value is intravenously injected pain orinjection often occurs. Where an antibody formulation is used as aninjection, it is desirable to have a pH value in an approximatelyneutral pH range, it is also advantageous to minimise surfactant levelsto avoid bubbles in the formulation which are detrimental for injectioninto subjects.

A liquid formulation of monoclonal anti-CTLA4 antibody is known fromWO2006/096491 (Pharmacia and Upjohn Company), and comprises 20 mg/mlantibody, 20 mM histidine buffer, 84 mg/ml trehelose, 0.2 mg/ml PS80surfactant, 0.05 mg/ml EDTA pH 5.5.

There is a need for a stable liquid antibody formulation which stablysupports high concentrations of bioactive antibody in solution and issuitable for parenteral administration, including intravenousintramuscular, intraperitoneal, intradermal or subcutaneous injection.It is further desirable that the formulation has minimised risk ofbubble formation and anaphylactoid side effects.

Furthermore there is a need to provide such a stable liquid formulationfor an anti-NGF antibody. NGF is known to play a central role in thedevelopment and maintenance of both peripheral and central neurons. Inaddition to its effects in the nervous system, increased NGF levels hasbeen linked to a variety of inflammatory conditions including systemiclupus erythematosus, multiple sclerosis, psoriasis, arthritis,interstitital cystitis and asthma. NGF also has a demonstrated activityin a variety of pain conditions. It has been shown that the anti-NGFantibody E3 is useful in the treatment of acute and chronic painconditions including, cancer pain, rheumatoid arthritis pain,osteoarthritis pain and post-surgical pain also (see for exampleWO2004/058184). There is a need for a stable liquid antibody preparationof an anti-NGF antibody to meet the medical need of patients sufferingfrom inflammatory and pain conditions mediated by NGF.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a liquid compositioncomprising; at least one antibody, at least one tonicity agent, at leastone buffer, at least one chelating agent, at least one surfactant,wherein the pH of said composition is from 5.0 to 7.5.

In one aspect, the present invention provides a liquid compositioncomprising; at least one antibody, at least one tonicity agent, at leastone buffer, at least one chelating agent, at least one surfactant,wherein the pH of said composition is from 5.8 to 6.8 The presentinvention also provides a liquid composition consisting of, orconsisting essentially of; at least one antibody, at least one tonicityagent, at least one buffer, at least one chelating agent, at least onesurfactant, wherein the pH of said composition is from 5.8 to 6.8.

The liquid composition according to the present invention provides theadvantages that it stably supports high concentrations of bioactiveantibody in solution and is suitable for parenteral administration,including intravenous, intramuscular, intraperitoneal, intradermal orsubcutaneous injection. Also it has minimised risk of bubble formationand anaphylactoid side effects.

According to a preferred embodiment of the present invention the liquidcomposition can comprise at least one antibody. In some embodiments,more than one antibody may be present. At least one, at least two, atleast three, at least four, at least five, or more, different antibodiescan be present. Generally, the two or more different antibodies havecomplementary activities that do not adversely affect each other.

The, or each, antibody can also be used in conjunction with other agentsthat serve to enhance and/or complement the effectiveness of theantibodies.

According to a preferred embodiment of the present invention the pH canbe in the range 5.0 to 7.5, more preferably between about pH 7.5 and ofany of about pH 5.1, 5.2, 5.3, 5.4 or 5.5. Further preferably the pH isin the range selected from between any one of pH 5.6, 5.7 or 5.8 and anyone of about pH 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5,6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8 or 5.7.

In a preferred embodiment the pH can be in the range of between about pH5.5 and any of about pH 6.0, 6.2, 6.5 or 6.8, alternatively the pH canbe in the range of between about pH 5.8 and any of about pH 6.0, 6.2,6.5 or 6.8.

More preferably the pH can be selected from pH values of any of about5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5, most preferably the pH is pH6.0+/−0.2. Values of pH in these ranges provides the liquid compositionwith enhanced protection from antibody aggregation and fragmentation andis close to physiological pH (about pH 7.2 to 7.4) for reduced risk ofpain or anaphylactoid side effects on injection.

According to a further preferred embodiment of the present invention thetonicity agent preferably comprises a polyol, a saccharide, acarbohydrate, a salt, such as sodium chloride, or mixtures thereof.Preferably the polyol has a molecular weight that is less than about 600kD (e.g., in the range from about 120 to about 400 kD), preferablyselected from mannitol, trehalose, sorbitol, erythritol, isomalt,lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol,polyethylene glycol, inositol, or mixtures thereof. Preferably thesaccharide or carbohydrate is selected from the group ofmonosaccharides, disaccharides and polysaccharides or mixtures thereof.Preferably the saccharide or carbohydrate is selected from the groupconsisting of fructose, glucose, mannose, sucrose, sorbose, xylose,lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrins,soluble starch, hydroxyethyl starch, water-soluble glucans, and mixturesthereof. Preferably the tonicity agent comprises a saccharide selectedfrom the group of reducing sugar or non reducing sugar or mixturesthereof. Further preferably the tonicity agent comprises a saccharidewhich is a non-reducing sugar, preferably selected from the groupconsisting of sucrose, trehalose, and mixtures thereof. Most preferablythe tonicity agent comprises trehalose, preferably trehalose dihydrate.According to the present invention the tonicity agent, particularlytrehalose, preferably trehalose dihydrate, provides the liquidcomposition with enhanced antibody stability and resistance toaggregation, oxidation and fragmentation during refrigerated storage,e.g. 0 to 10° C., particularly 5 to 8° C., more particularly 5° C., orfrozen storage and in cycles of freezing and thawing. Trehalose isparticularly advantageous as the resulting antibody formulation does notsuffer glycation.

The concentration of the tonicity agent in the liquid composition rangesfrom about 1 mg/ml to about 300 mg/ml, from about 1 mg/ml to about 200mg/ml, or from about 1 mg/ml to about 100 mg/ml. Preferably theconcentration of the tonicity agent in the liquid composition is about60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80mg/ml, about 81 mg/ml, about 82 mg/ml, about 83 mg/ml, about 84 mg/ml,about 85 mg/ml, about 86 mg/ml, about 87 mg/ml, about 88 mg/ml, about 89mg/ml, about 90 mg/ml, about 91 mg/ml, about 92 mg/ml, about 93 mg/ml,about 94 mg/ml, about 95 mg/ml, about 96 mg/ml, about 97 mg/ml, about 98mg/ml, about 99 mg/ml, about 100 mg/ml, about 105 mg/ml, about 110mg/ml, about 120 mg/ml, or about 130 mg/ml. Most preferably theconcentration of the tonicity agent in the liquid composition is about84 mg/ml.

Where the tonicity agent comprises a salt, the concentration of the saltin the liquid composition ranges from about 1 mg/ml to about 20 mg/ml.Salts that are pharmaceutically acceptable and suitable for thisinvention include sodium chloride, sodium succinate, sodium sulfate,potassium chloride, magnesium chloride, magnesium sulfate, and calciumchloride. Preferred salts for this invention are sodium chloride andmagnesium chloride, magnesium chloride may also improve the antibodystability by protecting the protein from deamidation. Preferably the thesalt in the liquid composition is selected from a range ofconcentrations of any of about 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5mg/ml, 6 mg/ml, 7 mg/ml, 8, mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19mg/ml and 20 mg/ml.

According to a preferred embodiment of the present invention thesurfactant is preferably selected from the group consisting ofpolysorbates, poloxamers, tritons, sodium dodecyl sulfate, sodium laurelsulfate, sodium octyl glycoside, lauryl-sulfobetaine,myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine,lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine,stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine,lauroamidopropyl-betaine, cocamidopropyl-betaine,linoleamidopropyl-betaine, myristamidopropyl-betaine,palmidopropyl-betaine, isostearamidopropyl-betaine,myristamidopropyl-dimethylamine, palmidopropyl-dimethylamine,isostearamidopropyl-dimethylamine, sodium methyl cocoyl-taurate,disodium methyl oleyl-taurate, dihydroxypropyl PEG 5 linoleammoniumchloride, polyethylene glycol, polypropylene glycol, and mixturesthereof. Further preferably the surfactant is selected from the groupconsisting of polysorbate 20, polysorbate 21, polysorbate 40,polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80,polysorbate 81, polysorbate 85, and mixtures thereof. More preferablythe surfactant is selected from polysorbate 20, polysorbate 80, PEG3350,or mixtures thereof. Most preferably the surfactant is polysorbate 20.According to the present invention the surfactant, particularlypolysorbate 20, provides the liquid composition with enhanced antibodystability and resistance to aggregation and fragmentation.

The concentration of the surfactant generally ranges from about 0.01mg/ml to about 10 mg/ml, from about 0.01 mg/ml to about 5.0 mg/ml, fromabout 0.01 mg/ml to about 2.0 mg/ml, from about 0.01 mg/ml to about 1.5mg/ml, from about 0.01 mg/ml to about 01.0 mg/ml, from about 0.01 mg/mlto about 0.5 mg/ml, from about 0.01 mg/ml to about 0.4 mg/ml, from about0.01 mg/ml to about 0.3 mg/ml, from about 0.01 mg/ml to about 0.2 mg/ml,from about 0.01 mg/ml to about 0.15 mg/ml, from about 0.01 mg/ml toabout 0.1 mg/ml, or from about 0.01 mg/ml, to about 0.05 mg/ml. Furtherpreferably the concentration of the surfactant is about 0.5 mg/ml, about0.05 mg/ml about 0.06 mg/ml about 0.07 mg/ml about 0.08 mg/ml about 0.09mg/ml about 0.1 mg/ml about 0.11 mg/ml about 0.12 mg/ml about 0.13 mg/mlabout 0.14 mg/ml about 0.15 mg/ml about 0.16 mg/ml about 0.17 mg/mlabout 0.18 mg/ml about 0.19 mg/ml, about 0.2 mg/ml. Most preferably theconcentration of the surfactant is about 0.1 mg/ml. Embodiments with theconcentration of the surfactant of about 0.1 mg/ml are highly preferredas this concentration permits maintenance of the stability of theantibody of the formulation in solution whilst also reducing thetendency for the formation of bubbles in the formulation duringpreparation of the formulation, handling of the formulation andpreparation for parenteral administration and especially from stressrelated to shaking and agitation during preparation and also duringshipping.

According to a preferred embodiment of the present invention the buffercan be selected from the group consisting of acetate, succinate,gluconate, citrate, histidine, acetic acid, phosphate, phosphoric acid,ascorbate, tartartic acid, maleic acid, glycine, lactate, lactic acid,ascorbic acid, imidazole, bicarbonate and carbonic acid, succinic acid,sodium benzoate, benzoic acid, gluconate, edetate, acetate, malate,imidazole, tris, phosphate, and mixtures thereof. Preferably the bufferis histidine, wherein the histidine can comprise either L-histidine orD-histidine, a solvated form of histidine, a hydrated form (e.g.,monohydrate) of histidine, or an anhydrous form of histidine or amixture thereof.

According to the present invention the buffer, particularly thepreferred buffer histidine, provides the liquid composition with a pHclose to physiological pH for reduced risk of pain or anaphylactoid sideeffects on injection and also provides enhanced antibody stability andresistance to aggregation, oxidation and fragmentation.

The concentration of the buffer can range from about 0.1 millimolar (mM)to about 100 mM. Preferably, the concentration of the buffer is fromabout 0.5 mM to about 50 mM, further preferably about 1 mM to about 30mM, more preferably about 1 mM to about 18 mM, increasingly preferablyabout 1 mM to about 15 mM. Preferably, the concentration of the bufferis about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM,about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about23 mM, about 24 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM,about 45 mM or about 50 mM. Most preferably the concentration of thebuffer is about 10 mM.

According to a preferred embodiment of the present invention thechelating agent can be selected from the group consisting of,aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substitutedglycines, 2-(2-amino-2-oxocthyl) aminoethane sulfonic acid (BES),deferoxamine (DEF), citric acid, niacinamide, and desoxycholates andmixtures thereof. Further preferably the chelating agent is selectedfrom the group consisting of ethylenediaminetetraacetic acid (EDTA),diethylenetriamine pentaacetic acid 5 (DTPA), nitrilotriacetic acid(NTA), N-2-acetamido-2-iminodiacetic acid (ADA),bis(aminoethyl)glycolether, N,N,N′,N′-tetraacetic acid (EGTA),trans-diaminocyclohexane tetraacetic acid (DCTA), glutamic acid, andaspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA),N,N-bis-hydroxyethylglycine (bicine) and N-(trishydroxymethylmethyl) 10glycine (tricine), glycylglycine, sodium desoxycholate, ethylenediamine;propylenediamine; diethylenetriamine; triethylenetetraamine (trien),ethylenediaminetetraaceto EDTA; disodium EDTA, calcium EDTA oxalic acid,malate, citric acid, citric acid monohydrate, and trisodiumcitrate-dihydrate, 8-hydroxyquinolate, amino acids, histidine, cysteine,methionine, peptides, polypeptides, and proteins and mixtures thereof.Further preferably the chelating agent is selected from the groupconsisting of salts of EDTA including dipotassium edetate, disodiumedetate, edetate calcium disodium, sodium edetate, trisodium edetate,and potassium edetate; and a suitable salt of deferoxamine (DEF) isdeferoxamine mesylate (DFM), or mixtures thereof. Chelating agents usedin the invention can be present, where possible, as the free acid orfree base form or salt form of the compound, also as an anhydrous,solvated or hydrated form of the compound or corresponding salt.

Most preferably the chelating agent is either disodium EDTA, calciumEDTA, most preferably disodium EDTA.

Particularly preferable is disodium EDTA as it provides the liquidcomposition with an enhanced antibody stability and/or resistance toaggregation.

The concentration of chelating agent generally ranges from about 0.01mg/ml to about 50 mg/ml, from about 1 mg/ml to about 10.0 mg/ml, fromabout 15 mg/ml to about 5.0 mg/ml, from about 0.01 mg/ml to about 1.0mg/ml, or from about 0.03 mg/ml to about 0.5 mg/ml. Further preferablyconcentration of chelating agent generally ranges from from about 0.01mM to about 2.0 mM, from about 0.01 mM to about 1.5 mM, from about 0.01mM to about 0.5 mM, from about 0.01 mM to about 0.4 mM, from about 0.01mM to about 0.3 mM, from about 0.01 mM to about 0.2 mM, from about 0.01mM to about 0.15 mM, from about 0.01 mM to about 0.1 mM, from about 0.01mM to about 0.09 mM, from about 0.01 mM to about 0.08 mM, from about0.01 mM to about 007 mM, from about 0.01 mM to about 0.06 mM, from about0.01 mM to about 0.05 mM, from about 0.01 mM to about 0.04 mM, fromabout 0.01 mM to about 0.03 mM, from about 0.01 mM to about 0.02 mM orfrom about 0.05 mM to about 0.01 mM. Preferably the concentration ofchelating agent can be about 0.01 mg/ml, 0.02 mg/ml, 0.03 mg/ml, about0.04 mg/ml, about 0.05 mg/ml, about 0.06 mg/ml, about 0.07 mg/ml, about0.10 mg/ml, about 0.20 mg/ml. Further preferably the concentration ofchelating agent is about 0.045 mg/ml, about 0.046 mg/ml, about 0.047mg/ml, about 0.048 mg/ml, about 0.049 mg/ml, about 0.05 mg/ml, about0.051 mg/ml, about 0.052 mg/ml, about 0.053 mg/ml, about 0.054 mg/ml,about 0.055 mg/ml, or about 0.056 mg/ml. Most preferably, theconcentration of chelating agent is about 0.05 mg/ml.

Chelating agents can lower the formation of reduced oxygen species,reduce acidic species (e.g., deamidation) formation, reduce antibodyaggregation, and/or reduce antibody fragmentation, and/or reduceantibody oxidation in the compositions of the present invention. Suchchelating agents can reduce or prevent degradation of an antibody thatis formulated in comparison to the antibody without the protection of achelating agent.

Unless stated otherwise, the concentrations listed herein are thoseconcentrations at ambient conditions, [i.e., at 25° C. and atmosphericpressure].

According to a preferred embodiment of the present invention the liquidcomposition can further comprise an antioxidant agent. Preferably theantioxidant is selected from the group comprising, methionine, sodiumthiosulfate, catalase, and platinum.

The concentration of antioxidant generally ranges from about 0.01 mg/mlto about 50 mg/ml, from about 0.01 mg/ml to about 10.0 mg/ml, from about0.01 mg/ml to about 5.0 mg/ml, from about 0.01 mg/ml to about 1.0 mg/ml,or from about 0.01 mg/ml to about 0.02 mg/ml. Preferably theconcentration of antioxidant can be about 0.01 mg/ml, 0.02 mg/ml, 0.03mg/ml, about 0.04 mg/ml, about 0.05 mg/ml, about 0.06 mg/ml, about 0.07mg/ml, 0.08 mg/ml, 0.09 mg/ml about 0.10 mg/ml, 0.11 mg/ml, 0.12 mg/ml,0.13 mg/ml, about 0.14 mg/ml, about 0.15 mg/ml, about 0.16 mg/ml, about0.17 mg/ml, 0.18 mg/ml, 0.19 mg/ml about 0.20 mg/ml, about 0.25 mg/ml,0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9mg/ml, 1.0 mg/ml. Most preferably, the concentration of antioxidant isabout 0.01 mg/ml.

According to a further preferred embodiment of the present invention theliquid composition can further comprise a preservative. Preferably thepreservative agent is selected from Phenol, m-cresol, benzyl alcohol,benzalkonium chloride, benzalthonium chloride, phenoxyethanol and methylparaben.

The concentration of preservative generally ranges from about 0.001mg/ml to about 50 mg/ml, from about 0.005 mg/ml to about 15.0 mg/ml,from about 0.008 mg/ml to about 12.0 mg/ml or from about 0.01 mg/ml toabout 10.0 mg/ml. Preferably the concentration of preservative can beabout 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, about 0.4 mg/ml, about 0.5 mg/ml,about 0.6 mg/ml, about 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml about 1.0 mg/ml,2.0 mg/ml, 3.0 mg/ml, about 4.0 mg/ml, about 5.0 mg/ml, about 6.0 mg/ml,about 7.0 mg/ml, 8.0 mg/ml, 9.0 mg/ml about 9.1 mg/ml, about 9.2 mg/ml,9.3 mg/ml, 9.4 mg/ml, 9.5 mg/ml, 9.6 mg/ml, 9.7 mg/ml, 9.8 mg/ml, 9.9mg/ml, 10.0 mg/ml. Most preferably, the concentration of preservative isabout 0.1 mg/ml or 9.0 mg/mL.

According to an aspect of the present invention the liquid formulationdoes not contain an antioxidant.

According to an aspect of the present invention the liquid formulationdoes not contain a preservative.

According to preferred embodiment of the present invention the presentinvention the concentration of antibody can range from about 0.1 toabout 200 mg/ml.

Preferably the concentration of antibody is about 0.5 mg/ml, about 1mg/ml, about 2 mg/ml, about 2.5 mg/ml, about 3 mg/ml, about 3.5 mg/ml,about 4 mg/ml, about 4.5 mg/ml, about 5 mg/ml, about 5.5 mg/ml, about 6mg/ml, about 6.5 mg/ml, about 7 mg/ml, about 7.5 mg/ml, about 8 mg/ml,about 8.5 mg/ml, about 9 mg/ml, about 9.5 mg/ml, about 10 mg/ml, about11 mg/ml, about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15mg/ml, about 16 mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml,about 20 mg/ml, about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24mg/ml, about 25 mg/ml, about 26 mg/ml, about 27 mg/ml, about 28 mg/ml,about 29 mg/ml, about 30 mg/ml, about 31 mg/ml, about 32 mg/ml, about 33mg/ml, about 34 mg/ml, about 35 mg/ml, about 36 mg/ml, about 37 mg/ml,about 38 mg/ml, about 39 mg/ml, about 40 mg/ml, about 41 mg/ml, about 42mg/ml, about 43 mg/ml, about 44 mg/ml, about 45 mg/ml, about 46 mg/ml,about 47 mg/ml, about 48 mg/ml, about 49 mg/ml, about 50 mg/ml, about 51mg/ml, about 52 mg/ml, about 53 mg/ml, about 54 mg/ml, about 55 mg/ml,about 56 mg/ml, about 57 mg/ml, about 58 mg/ml, about 59 mg/ml, about 60mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/mlor about 110 mg/ml. Most preferably the concentration of antibody isless than or equal to about 50 mg/ml and may be selected from the groupcomprising about 2 mg/ml, about 2.5 mg/ml, about 5 mg/ml, about 10mg/ml, about 19 mg/ml, about 20 mg/ml, 22 mg/ml and about 50 mg/ml.

The antibody is preferably selected from the group of; monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv, Fc, ScFv etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion (e.g., a domain antibody),humanized antibodies, human antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site of the required specificity, including glycosylationvariants of antibodies, amino acid sequence variants of antibodies, andcovalently modified antibodies. The antibody may be murine, rat, human,or any other origin (including chimeric or humanized antibodies). Insome embodiments, the antibody can be human but is more preferablyhumanized. Preferably the antibody is isolated, further preferably it issubstantially pure. Where the antibody is an antibody fragment thispreferably retains the functional characteristics of the originalantibody i.e. the ligand binding and/or antagonist or agonist activity.

According to a preferred embodiment of the present invention theantibody heavy chain constant region may be from any type of constantregion, such as IgG, IgM, IgD, IgA, and IgE; and any isotypes, such asIgGI, IgG2, IgG3, and IgG4. Preferably the antibody is an IgG2 antibody.

According to the present invention, the antibody can comprise the humanheavy chain IgG2a constant region. In some embodiments the antibodycomprises the human light chain kappa constant region. In someembodiments, the antibody comprises a modified constant region, such asa constant region that is immunologically inert, e.g., does not triggercomplement mediated lysis, or does not stimulate antibody-dependent cellmediated cytotoxicity (ADCC). In other embodiments, the constant regionis modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCTpublication No. WO099/58572; and/or UK Patent Application No. 9809951.8.In still other embodiments, the antibody comprises a human heavy chainIgG2a constant region comprising the following mutations: A330P331 toS330S331 (amino acid numbering with reference to the wildtype IgG2asequence), Eur. J. Immunol. (1999) 29:2613-2624.

According to a preferred embodiment of the present invention, theantibody is an anti-NGF antibody that binds NGF (such as human NGF) witha high affinity. In some embodiments, high affinity is (a) binding NGFwith a K_(D) of less than about 2 nM (such as any of about 1 nM, 800 pM,600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30 pM, 20 pM, 10 pM, 5 pM or less), and/or a k_(off) of slower thanabout 6×10⁻⁵ s⁻¹; and/or (b) inhibiting, (reducing, and/or blocking)human NGF-dependent survival of mouse E13.5 trigeminal neurons with anIC50 (in the presence of about 15 pM of NGF) of any of about 200 pM, 150pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM, 10 pM, or less; and/or (c)inhibiting (reducing, and/or blocking) human NGF-dependent survival ofmouse E13.5 trigeminal neurons with an IC50 (in the presence of about1.5 pM of NGF) of any of about 50 pM, 40 pM, 30 pM, 10 pM, 20 pM, 10 pM,5 pM, 2 pM, 1 pM, or less; and/or (d) inhibiting (reducing, and/orblocking) rat NGF-dependent survival of mouse E13.5 trigeminal neuronswith an IC50 (in the presence of about 15 pM of NGF) of any of about 150pM, 125 pM, 100 pM, 80 pM, 60 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, orless; and/or (e) inhibiting (reducing, and/or blocking) ratNGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50(in the presence of about 1.5 pM of NGF) of any of about 30 pM, 25 pM,20 pM, 15 pM, 10 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, or less; and/or (f)and/or bind NGF with higher affinity than does the trkA receptor.

In another aspect, the antibodies (a) bind NGF (such as human NGF) witha K_(D) of less than about 2 nM (such as any of about 1 nM, 800 pM, 600pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30pM, 20 pM, 10 pM, 5 pM or less), and/or a k_(off) of slower than about6×10⁻⁵ s⁻¹; and/or (b) inhibit human NGF-dependent survival of mouseE13.5 trigeminal neurons with an IC50 (in the presence of about 15 pM ofNGF) of any of about 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM,10 pM, or less; and/or (c) inhibit human NGF-dependent survival of mouseE13.5 trigeminal neurons with an IC50 (in the presence of about 1.5 pMof NGF) of any of about 50 pM, 40 pM, 30 pM, 10 pM, 20 pM, 10 pM, 5 pM,2 pM, 1 pM, or less; and/or bind NGF with higher affinity than does thetrkA receptor. In some embodiments, the antibodies (a) bind NGF with aK_(D) of less than about 2 nM; and/or (b) inhibit human NGF-dependentsurvival of mouse E13.5 trigeminal neurons with an IC50 of about 100 pMor less, wherein the IC50 is measured in the presence of about 15 pMNGF; and/or (c) inhibit human NGF-dependent survival of mouse E13.5trigeminal neurons with an IC50 of about 10 pM or less, wherein the IC50is measured in the presence of about 1.5 pM of NGF. In some embodiments,the antibodies (a) bind NGF with a K_(D) of less than about 100 pM;and/or (b) inhibit human NGF-dependent survival of mouse E13.5trigeminal neurons with an IC50 of about 20 pM or less, wherein the IC50is measured in the presence of about 15 pM NGF; and/or (c) inhibit humanNGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 ofabout 2 pM or less, wherein the IC50 is measured in the presence ofabout 1.5 pM of NGF.

The epitope (s) that can be bound by the antibody can be continuous ordiscontinuous. In one embodiment, the antibody binds essentially thesame hNGF epitopes as an antibody selected from the group consisting ofMAb 911, MAb 912, and MAb 938 as described in Hongo et al., Hybridoma,19: 215-227 (2000), an antibody defined herein (such as antibody E3);and/or described in WO2005019266 (including antibodies 4D4, 14D10, 6G9,7H2, 14F11 and 4G6) or WO2006131951 (including antibody Hu-aD11),WO09023540 or US20090041717, the entire content of which are hereinincorporated by reference. In another embodiment, the antibody bindsessentially the same hNGF epitope as MAb 911. In still anotherembodiment, the antibody binds essentially the same epitope as MAb 909.Hongo et al., supra. For example, the epitope may comprise one or moreof residues K32, K34 and E35 within variable region 1 (amino acids23-35) of hNGF; residues F79 and T81 within variable region 4 (aminoacids 81-88) of hNGF; residues H84 and K88 within variable region 4;residue R103 between variable region 5 (amino acids 94-98) of hNGF andthe C-terminus (amino acids 111-118) of hNGF; residue E11 withinpre-variable region 1 (amino acids 10-23) of hNGF; Y52 between variableregion 2 (amino acids 40-49) of hNGF and variable region 3 (amino acids59-66) of hNGF; residues L112 and S113 within the C-terminus of hNGF;residues R59 and R69 within variable region 3 of hNGF; or residues V18,V20, and G23 within pre-variable region 1 of hNGF. In addition, anepitope can comprise one or more of the variable region 1, variableregion 3, variable region 4, variable region 5, the N-terminus region,and/or the C-terminus of hNGF. In still another embodiment, the antibodysignificantly reduces the solvent accessibility of residue R103 of hNGF.It is understood that although the epitopes described above relate tohuman NGF, one of ordinary skill can align the structures of human NGFwith the NGF of other species and identify likely counterparts to theseepitopes.

In one aspect, the invention provides polypeptides (such as anantibody), which comprise a heavy chain variable region comprising SEQID NO: 9, wherein I34 is S, L, V A, or I; and N35 is substituted with N,T or S. For convenience herein, “substituted” or “is” in this context orreference to an amino acid refers to choices of amino acid (s) for agiven position. As is clear, the substitution, or choice, may be theamino acid depicted in a SEQ ID or Figure. Residue numbers aredetermined readily from reference to the SEQ ID NO stated and follow theresidue numbering of the antibody.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a heavy chain variable region comprising SEQ IDNO: 10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is Lor V.

In another aspect, the invention provides polypeptides (such as anantibody) which comprises a heavy chain variable region comprising SEQID NO: 11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W; whereinG103 is G, A, or S; wherein T104 is T or S; wherein S 105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D 109 is D, N, or G; and wherein Y110 is Y, K, S, R or T.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a heavy chain variable region comprising SEQ IDNO: 11, wherein Y 100 is Y, L, or R; wherein Y101 is Y or W; whereinG103 is G, A, or S; wherein T104 is T or S; wherein S 105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Y110 is anyamino acid.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a heavy chain variable region comprising SEQ IDNO: 11, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S,A, C, V, N, D, or T; wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S 105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; and whereinY110 is any amino acid.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a light chain variable region comprising SEQ IDNO: 12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, orQ.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a light chain variable region comprising SEQ IDNO: 13, wherein I51 is I, T, V or A; and S56 is S or T.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a light chain variable region comprising SEQ IDNO: 14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 isY or R.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a light chain variable region comprising SEQ IDNO: 14, wherein S91 is S or E; K92 is any amino acid; T93 is any aminoacid; and wherein Y96 is Y or R.

In one aspect, the invention provides polypeptides (such as anantibody), which comprise an amino acid sequence shown in SEQ ID NO: 9,wherein I34 is S, L, V A, or I; and N35 is N, T or S.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 10,wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G,A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is D, N, or G; and wherein Y110 is Y, K, S, R or T.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G,A, or S; wherein T104 is T or S; wherein S 105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is S, A, C, G, D, N, T, or G; and wherein Y110 is any aminoacid.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 11,wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S, A, C, V,N, D, or T; wherein Y100 is Y, L, or R; wherein Y101 is Y or W; whereinG103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Y110 is anyamino acid.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 12,wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 13,wherein I51 is I, T, V or A; and S56 is S or T.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 14,wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 14,wherein S91 is S or E; K92 is any amino acid; T93 is any amino acid; andwherein Y96 is Y or R.

In another aspect, the invention provides polypeptides (such anantibodies, including humanized antibodies) which comprise a heavy chainvariable region comprising the CDR1 region of SEQ ID NO: 9, wherein I34is S, L, V A, or I; and N35 is N, T or S; the CDR2 region of SEQ ID NO:10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L orV; and the CDR3 region of SEQ ID NO: 11, wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G;wherein Y110 is Y, K, S, R or T. In some embodiments, the heavy chainvariable region comprises the CDR3 region of SEQ ID NO: 11, wherein Y100is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G, A, or S;wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y,R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109is S, A, C, G, D, N, T, or G; wherein Y110 is any amino acid. In otherembodiments, the heavy chain variable region comprises the CDR3 regionof SEQ ID NO: 11, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R; wherein Y101is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; whereinS105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y orF; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G;and wherein Y110 is any amino acid. In some embodiments, the polypeptide(such as an antibody) further comprises an antibody light chain variableregion.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a light chain variable region comprising theCDR1 region of SEQ ID NO: 12, wherein S26 is S or F; D28 is D, S, A, orY; and H32 is H, N, or Q; the CDR2 region of SEQ ID NO: 13, wherein I51is I, T, V or A; and S56 is S or T; and the CDR3 region of SEQ ID NO:14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y orR. In some embodiments, the light chain variable region comprises theCDR3 region of SEQ ID NO: 14, wherein S91 is S or E; K92 is any aminoacid; T93 is any amino acid; and wherein Y96 is Y or R. In someembodiments, the polypeptide (such as an antibody) further comprises anantibody heavy chain.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise (a) a heavy chain variable region comprisingthe CDR1 region of SEQ ID NO: 9, wherein I34 is S, L, V A, or I; and N35is N, T or S; the CDR2 region of SEQ ID NO: 10, wherein M50 is M, I, G,Q, S, or L; A62 is A, or S; and L63 is L or V; and the CDR3 region ofSEQ ID NO: 11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is D, N, or G; wherein Y110 is Y, K, S, Ror T; and (b) a light chain variable region comprising the CDR1 regionof SEQ ID NO: 12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32is H, N, or Q; the CDR2 region of SEQ ID NO: 13, wherein I51 is I, T, Vor A; and S56 is S or T; and the CDR3 region of SEQ ID NO: 14, whereinS91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R. In someembodiments, the light chain variable region comprises the CDR3 regionof SEQ ID NO: 14, wherein S91 is S or E; K92 is any amino acid; T93 isany amino acid; and wherein Y96 is Y or R. In some embodiments, theheavy chain variable region comprises the CDR3 region of SEQ ID NO: 11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G,A, or S; wherein T104 is T or S; wherein S 105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is S, A, C, G, D, N, T, or G; wherein Y110 is any aminoacid. In other embodiments, the heavy chain variable region comprisesthe CDR3 region of SEQ ID NO: 11, wherein G98 is G, S, A, C, V, N, D, orT; wherein G99 is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D,N, T, or G; and wherein Y110 is any amino acid. In some embodiments, thepolypeptide further comprises an antibody light chain.

In another aspect, the invention provides polypeptides (such anantibody, including a humanized antibody) which comprise an amino acidsequence shown in SEQ ID NO: 9, wherein I34 is S, L, V A, or I; and N35is N, T or S; an amino acid sequence shown in SEQ ID NO: 10, wherein M50is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and an aminoacid sequence shown in SEQ ID NO: 11, wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G;wherein Y110 is Y, K, S, R or T. In some embodiments, the polypeptidecomprises an amino acid sequence shown in SEQ ID NO: 11, wherein Y100 isY, L, or R; and wherein Y101 is Y or W; wherein G103 is G, A, or S;wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y,R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109is S, A, C, G, D, N, T, or G; and wherein Y110 is any amino acid. Inother embodiments, the polypeptide comprises an amino acid sequenceshown in SEQ ID NO: 11, wherein G98 is G, S, A, C, V, N, D, or T;wherein G99 is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D,N, T, or G; and wherein Y110 is any amino acid. In some embodiments, thepolypeptide (such as an antibody) further comprises an antibody lightchain variable region.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 12,wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q; anamino acid sequence shown in SEQ ID NO: 13, wherein I51 is I, T, V or A;and S56 is S or T; and an amino acid sequence shown in SEQ ID NO: 14,wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R.

According to a further preferred embodiment of the present invention theantibody can be an anti-NGF antibody comprising a heavy chain variableregion comprising:

(a) a CDR1 region shown in SEQ ID NO: 3;

(b) a CDR2 region shown in SEQ ID NO:4; and

(c) a CDR3 region shown in SEQ ID NO:5, 11, 56, 58, 60, 62, 64.

According to a further preferred embodiment of the present invention theantibody can be anti-NGF antibody comprising a light chain variableregion comprising:

(a) a CDR1 region shown in SEQ ID NO: 6;

(b) a CDR2 region shown in SEQ ID NO:7; and

(c) a CDR3 region shown in SEQ ID NO:8, 14, 55, 57, 59, 61 or 63.

According to a preferred embodiment of the present invention theantibody can be an anti-NGF antibody comprising a heavy chain variableregion comprising:

(a) a CDR1 region shown in SEQ ID NO: 3;

(b) a CDR2 region shown in SEQ ID NO:4; and

(c) a CDR3 region shown in SEQ ID NO:5.

According to the present invention the antibody can be anti-NGF antibodycomprising a light chain variable region comprising:

(a) a CDR1 region shown in SEQ ID NO: 6;

(b) a CDR2 region shown in SEQ ID NO:7; and

(c) a CDR3 region shown in SEQ ID NO:8.

The anti-NGF antibody may further comprise a heavy chain variable regioncomprising:

(a) a CDR1 region shown in SEQ ID NO: 3;

(b) a CDR2 region shown in SEQ ID NO:4; and

(c) a CDR3 region shown in SEQ ID NO:5.

The anti-NGF antibody may comprise a heavy chain variable regioncomprising an amino acid sequence of any of at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID No. 1 and/or a light chain variable regioncomprising an amino acid sequence of any of at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID No. 2, wherein the antibody bindsspecifically to NGF.

The heavy chain variable region and/or light chain variable region of ahumanised anti-NGF antibody may comprise one or more respectiveframework mutations. In one aspect the framework mutation may replace ahuman framework residue with the complementary mouse framework residue.The mutation may comprise the substitution V71 K in the heavy chainvariable region.

The anti-NGF antibody may comprise a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 1 and/or may comprise alight chain variable region comprising the amino acid sequence of SEQ IDNO: 2.

The anti-NGF antibody may be an antibody comprising the amino acidsequences shown in SEQ ID NOS: 1 and 2.

The anti-NGF antibody may comprise a heavy chain region comprising anamino acid sequence of any of at least about 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID No. 16 and/or a light chain region comprising an amino acidsequence of any of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ IDNo. 17, wherein the antibody binds specifically to NGF.

The anti-NGF antibody may comprise a heavy chain region comprising theamino acid sequence of SEQ ID NO: 16 and/or may comprise a light chainregion comprising the amino acid sequence of SEQ ID NO: 17.

The anti-NGF antibody may be an antibody comprising the amino acidsequences shown in SEQ ID NOS: 16 and 17.

The anti-NGF antibody may compete for NGF binding with an anti-NGFantibody as defined herein. The anti-NGF antibody may compete for NGFbinding with an antibody comprising a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 1 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 2.

The anti-NGF antibody may be a humanized and affinity matured antibody,E3, which specifically binds human and rodent NGF. Antibody E3 isdescribed in WO2004/058184, the content of which is hereby incorporatedby reference in its entirety. The amino acid sequences of the heavychain and light chain variable regions of E3 are shown in SEQ ID Nos. 1and 2 (FIGS. 1A and 1B of WO2004/058184), respectively. The CDR portionsof antibody E3 (including Chothia and Kabat CDRs) are diagrammaticallydepicted in FIGS. 1A and 1B of WO2004/058184. The amino acid sequencesof E3 heavy and light chains, and of the individual extended CDRs arealso shown below (See, “antibody sequences”, below). Antibody E3 ishighly potent in sequestering NGF and preventing interaction with itsreceptor. E3 and its murine precursor antibody 911 have been shown to bean effective analgesic in non-clinical animal models of pathologicalpain including arthritis, cancer pain and post surgical pain.

The anti-NGF antibody may also comprise a fragment or a region of theantibody E3 (interchangeably termed “E3” herein). In one embodiment, thefragment is a light chain of the antibody E3 as shown in FIG. 1B ofWO2004/058184 and SEQ ID No. 17 herein. In another embodiment, thefragment is a heavy chain of the antibody E3 as shown in FIG. 1A ofWO2004/058184 and SEQ ID No. 16 herein. In yet another embodiment, thefragment contains one or more variable regions from a light chain and/ora heavy chain of the antibody E3. In yet another embodiment, thefragment contains one or more CDRs from a light chain and/or a heavychain of the antibody E3 as shown in FIGS. 1A and 1B of WO2004/058184and SEQ ID Nos. 17 and 16, respectively, herein.

In another aspect, the antibody comprises a light chain that is encodedby a polynucleotide that is produced by a host cell with a depositnumber of ATCC No.

PTA-4893 or ATCC No. PTA-4894. In another aspect, the antibody comprisesa heavy chain that is encoded by a polynucleotide that is produced by ahost cell with a deposit number of ATCC No. PTA-4895. In another aspect,the antibody comprises (a) a light chain that is encoded by apolynucleotide that is produced by a host cell with a deposit number ofATCC No. PTA-4894 or ATCC No. PTA-4893; and (b) a heavy chain that isencoded by a polynucleotide that is produced by a host cell with adeposit number of ATCC No. PTA-4895 (for convenience herein, thepolynucleotide(s) produced by a deposited host cell are referred to ashaving a deposit number of ATCC NOs PTA-4894, PTA-4893 and PTA-4895). Inanother aspect, the antibody comprises a light chain variable region ofa light chain that is encoded by a polynucleotide that is produced by ahost cell with a deposit number of ATCC No. PTA-4894 or ATCC No.PTA-4893. In another aspect, the antibody comprises a heavy chainvariable region of a heavy chain that that is encoded by apolynucleotide that is produced by a host cell with a deposit number ofATCC No. PTA-4895. In another aspect, the antibody comprises (a) a lightchain variable region of a light chain that is encoded by apolynucleotide that is produced by a host cell with a deposit number ofATCC No. PTA-4894 or ATCC No. PTA-4893, and (b) a heavy chain variableregion of a heavy chain that that is encoded by a polynucleotide that isproduced by a host cell with a deposit number of ATCC No. PTA-4895. Instill another aspect, the antibody comprises one or more CDR(s) encodedby (a) a polynucleotide that is produced by a host cell with a depositnumber of ATCC No. PTA-4894; and/or (b) a heavy chain that is encoded bya polynucleotide that is produced by a host cell with a deposit numberof ATCC No. PTA-4895.

In another aspect, the antibody comprises any one or more of thefollowing: a) one or more CDR(s) of antibody E3 shown in SEQ ID Nos. 1-8or SEQ ID Nos. 3-8 (FIGS. 1A and 1B of WO2004/058184); b) CDR H3 fromthe heavy chain of antibody E3 shown in SEQ ID No. 1 and 5 (FIG. 1A ofWO2004/058184); c) CDR L3 from the light chain of antibody E3 shown inSEQ ID No. 2 and 8 (FIG. 1B of WO2004/058184); d) three CDRs from thelight chain of antibody E3 shown in SEQ ID No. 2, 6-8 (FIG. 1B ofWO2004/058184); e) three CDRs from the heavy chain of antibody E3 shownin SEQ ID Nos. 1, 3-5 (FIG. 1A of WO2004/058184); and f) three CDRs fromthe light chain and three CDRs from the heavy chain, of antibody E3shown in SEQ ID Nos. 1-8 (FIGS. 1A and 1B of WO2004/058184). In anotheraspect, the antibody may comprise any one or more of the following: a)one or more (one, two, three, four, five, or six) CDR(s) derived fromantibody E3 shown in SEQ ID Nos. 1-8 (FIGS. 1A and 1B of WO2004/058184);b) a CDR derived from CDR H3 from the heavy chain of antibody E3 shownin SEQ ID Nos. 1 and 5 (FIG. 1A of WO2004/058184); and/or c) a CDRderived from CDR L3 from the light chain of antibody E3 shown in SEQ IDNos. 2 and 8 (FIG. 1B of WO2004/058184). In some embodiments, the CDRsmay be Kabat CDRs, Chothia CDRs, or a combination of Kabat and ChothiaCDRs (termed “extended” or “combined” CDRs herein). In some embodiments,the polypeptides comprise any of the CDR configurations (includingcombinations, variants, etc.) described herein.

In another embodiment the antibody may comprise:

(a) a heavy chain variable region comprising:

(i) a CDR1 region of SEQ ID NO: 30;

(ii) a CDR2 region comprising the sequence of SEQ ID NO: 31;

(iii) a CDR3 region selected from the group consisting of SEQ ID NO: 11,56, 58, 60, 62 and 64; and

(b) a light chain variable region comprising:

(i) a CDR1 region of SEQ ID NO: 18;

(ii) a CDR2 region of SEQ ID NO: 19;

(iii) a CDR3 region selected from the group consisting of SEQ ID NO: 14,55, 57, 59, 61 and 63.

In some embodiments of the present invention the C-terminal lysine ofthe heavy chain of any of the anti-NGF antibodies described herein isdeleted. In various cases the heavy and/or light chain of the anti-NGFantibodies described herein may optionally include a signal sequence.

In another embodiment, the antibody may be selected from an anti-NGFantibody known in the art, such as antibodies described in WO2005019266(including antibodies 4D4, 14D10, 6G9, 7H2, 14F11 and 4G6), WO2006131951(including antibody Hu-aD11), WO09023540 or US20090041417. The antibodymay bind to the same epitope as an anti-NGF antibody known in the art,such as Mab911, MAb 912, and MAb 938 as described in Hongo et al.,Hybridoma, 19: 215-227 (2000), and antibodies described in WO2005019266(including antibodies 4D4, 14D10, 6G9, 7H2, 14F11 and 4G6), WO2006131951(including antibody Hu-aD11), WO09023540 or US20090041417 and/or maycompete for binding to NGF with such an antibody.

According to a further aspect of the present invention there is provideda liquid composition comprising or consisting of;

about 0.5 mg/ml to about 50 mg/ml of at least one antibody,

about 1.0 mM to about 15 mM histidine buffer,

about 1 mg/ml to about 100 mg/ml trehalose dihydrate,

about 0.01 to about 0.15 mg/ml PS20,

about 0.01 to about 0.1 mg/ml. disodium EDTA,

wherein said composition is of a pH selected from the the range ofbetween about pH 5.5 and any of about pH 6.0, 6.2, 6.5 or 6.8, oralternatively from the range of between about pH 5.8 and any of about pH6.0, 6.2, 6.5 or 6.8.

According to a further aspect of the present invention there is provideda liquid composition comprising or consisting of;

any of about 2.5 mg/ml, 5 mg/ml, 10 mg/ml, 20 mg/ml, 22 mg/ml or about50 mg/ml of at least one antibody,

about 1.0 mM to about 15 mM histidine buffer,

about 1 mg/ml to about 100 mg/ml trehalose dihydrate,

about 0.01 to about 0.15 mg/ml PS20,

about 0.01 to about 0.1 mg/ml. disodium EDTA,

wherein said composition is of a pH selected from the range of betweenabout pH 5.5 and any of about pH 6.0, 6.2, 6.5 or 6.8, or alternativelyfrom the range of between about pH 5.8 and any of about pH 6.0, 6.2, 6.5or 6.8.

According to a preferred embodiment the liquid composition comprises orconsists of;

any of about 2.5 mg/ml, 5 mg/ml, 10 mg/ml, 20 mg/ml, 22 mg/ml or about50 mg/ml of at least one antibody,

about 10 mM histidine buffer,

about 84 mg/ml trehalose dihydrate,

about 0.1 mg/ml PS20,

about 0.05 mg/ml. disodium EDTA,

wherein said composition is of a pH selected from the range of betweenabout pH 5.5 and any of about pH 6.0, 6.2, 6.5 or 6.8, or alternativelyfrom the range of between about pH 5.8 and any of about pH 6.0, 6.2, 6.5or 6.8, preferably is of a pH from 5.8 to 6.5, and wherein said antibodycomprises a variable heavy chain sequence of SEQ ID NO. 1 and a variablelight chain sequence of SEQ ID NO. 2.

According to a preferred embodiment the liquid composition comprises orconsists of;

any of about 2.5 mg/ml, 5 mg/ml, 10 mg/ml, 20 mg/ml, 22 mg/ml or about50 mg/ml of at least one antibody,

about 10 mM histidine buffer,

about 84 mg/ml trehalose dihydrate,

about 0.1 mg/ml PS20,

about 0.05 mg/ml. disodium EDTA,

wherein said composition is of pH 6.0, +/−0.2 and wherein said antibodycomprises a variable heavy chain sequence of SEQ ID NO. 1 and a variablelight chain sequence of SEQ ID NO. 2. In a preferred embodiment the dosevolume used is 1 ml.

In one aspect there is provided a liquid composition which is notlyophilized and has not been subjected to lyophylization.

According to a further aspect of the present invention there is provideda liquid composition of the invention wherein the composition isresistant to aggregation of the antibody after multiple freeze thawcycles.

Preferably the antibody is resistant to aggregation during at least onecycle of freezing and thawing of the composition, further preferablywherein the antibody is resistant to aggregation during multiple freezethaw cycles, preferably during at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 cycles of freeze thawing. Most preferably the antibodyis resistant to aggregation during at least fifteen cycles of freezingand thawing of the liquid composition, further preferably four orfifteen cycles.

Further preferably the antibody demonstrates less than about 10%aggregation increase after the multiple freeze thaw cycles of the liquidcomposition in comparison to the same composition, or an equivalentsample of identical or about identical composition, prior to freezethawing, more preferably less than or about, 9, 8, 7, 6, 5, 4, 3, 2, 1,0.5 or 0% aggregation increase after the multiple freeze thaw cycles ofthe liquid composition in comparison to the same composition, or anequivalent sample of identical or about identical composition, prior tofreeze thawing.

According to a further aspect of the present invention there is provideda liquid composition of the invention wherein the composition can bestored for a period of at least about 26 or 52 weeks at a temperature ofany of about 5, 25 or 40° C. and wherein there is less than about 35%further preferably less than about 10% increase in aggregation of theantibody of the composition. Preferably the concentration of theantibody is either about 10 mg/ml or about 50 mg/ml.

Preferably there is less than about 35% further preferably less thanabout 10% increase in aggregation of the antibody of the liquidformulation when stored for a period of any of about 2, 4, 8, 9, 13, 26or 52 weeks at a temperature of any of about 5, 25 or 40° C. incomparison to the same composition, or an equivalent sample of identicalor about identical composition, prior to the period of storage.Preferably the concentration of the antibody is either about 10 mg/ml orabout 50 mg/ml.

Further preferably the antibody of the liquid composition demonstratesless than about 35% further preferably less than about 10% aggregationincrease, further preferably less than any of about 34%, 33%, 32%, 31%,30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, more preferably less than about8, 7, 6, 5, 4, 3, 2, 1, or 0.5% or equal to about 0% aggregationincrease after the period of storage of 2, 4, 8, 9, 13, 26 or 52 weeksat a temperature of any of about 5, 25 or 40° C.; most preferably lessthan about 20% at 40° C. for 52 weeks, or less than about 10% at 40° C.for 26 weeks, in comparison to the same composition, or an equivalentsample of identical or about identical composition, prior to the periodof storage. Preferably the concentration of the antibody is either about10 mg/ml or about 50 mg/ml.

According to a further aspect of the present invention there is provideda liquid composition of the invention wherein the composition can bestored for a period of at least about 26 or 52 weeks at a temperatureany of about 5, 25 or 40° C. and wherein there is less than about 35%further preferably less than about 10% increase in oxidation of theantibody of the composition in comparison to the same composition, or anequivalent sample of identical or about identical composition, prior tothe period of storage. Preferably the concentration of the antibody iseither about 10 mg/ml or about 50 mg/ml.

Preferably the antibody of the liquid composition demonstrates less thanabout 35% further preferably less than about 10% oxidation increase,further preferably less than any of about 34%, 33%, 32%, 31%, 30%, 29%,28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, more preferably less than about 8, 7, 6, 5,4, 3, 2, 1 or 0.5% or equal to about 0% oxidation increase after theperiod of storage of any of about 2, 4, 8, 9, 13, 26 or 52 weeks at atemperature of any of about 5, 25 or 40° C., most preferably less thanabout 31% or 30% for 52 weeks, or less than about 11% or 10% at 40° C.for 26 weeks, in comparison to the same composition, or an equivalentsample of identical or about identical composition, prior to the periodof storage. Preferably the concentration of the antibody is either about10 mg/ml or about 50 mg/ml.

According to a further aspect of the present invention there is provideda liquid composition of the invention wherein the composition can bestored for a period of at least about 26 or 52 weeks at a temperature ofany of about 5, 25 or 40° C. and wherein there is less than 30% decreasein activity of the antibody of the composition.

Preferably there is less than about 30% decrease in activity of theantibody of the liquid formulation, further preferably less than about25%, further preferably 20% more preferably less than about 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 or 0%decrease in activity of the antibody of the formulation when stored fora period of any of at least about 2, 4, 8, 13, or 26 weeks at atemperature of any of about 5, 25 or 40° C. in comparison to the samecomposition, or an equivalent sample of identical or about identicalcomposition, prior to the period of storage. Preferably theconcentration of the antibody is either about 10 mg/ml or about 50mg/ml.

According to a further aspect of the present invention there is provideda liquid composition of the invention wherein the composition can bestored for a period of at least about 26 weeks at a temperature any ofabout 5, 25 or 40° C. and wherein there is less than about 15% increasein fragmentation of the antibody of the composition in comparison to thesame composition, or an equivalent sample of identical or aboutidentical composition, prior to the period of storage, preferablywherein the concentration of the antibody is either about 10 mg/ml orabout 50 mg/ml. Preferably the antibody of the liquid compositiondemonstrates less than about 15% further preferably further preferablyless than any of about 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, 1% or 0.5% or equal to about 0% fragmentation increase after theperiod of storage of any of about 2, 4, 8, 9, 13 or 26 weeks at atemperature of any of about 5, 25 or 40° C., most preferably less thanabout 14% at about 40° C. for about 26 weeks, in comparison to the samecomposition, or an equivalent sample of identical or about identicalcomposition, prior to the period of storage. Preferably theconcentration of the antibody is either about 10 mg/ml or about 50mg/ml.

According to a further aspect of the present invention there is provideda liquid composition of the invention wherein the composition can storedfor a period of at least about 52 weeks at a temperature of between 2 to8° C., preferably 5° C. wherein there is less than 20% decrease inactivity of the antibody of the composition. Preferably theconcentration of antibody is any of about 2.5, 5, 10, 20 or 50 mg/ml.

Preferably there is less than about 15%, further preferably less thanabout 10% decrease in activity of the antibody of the liquid formulationwhen stored for a period of any of at least about 2, 4, 8, 13, 26 or 52weeks, further preferably any of at least about 2, 3, 6, 9, 12, 18 or 24months at a temperature of about 2 to 8° C., preferably 5° C. incomparison to the same composition, an equivalent sample of identical orabout identical composition, prior to the period of storage. Preferablythe concentration of antibody is any of about 2.5, 5, 10, 20 or 50mg/ml.

Further preferably the antibody of the liquid composition demonstratesless than about 15% aggregation increase, further preferably less thanabout 10%, more preferably less than about 9, 8, 7, 6, 5, 4, 3, 2, 1,0.5 or 0% activity decrease after the period of storage in comparison tothe same composition, or an equivalent sample of identical or aboutidentical composition, prior to the period of storage.

According to a further aspect of the present invention there is provideda liquid composition of the invention wherein the composition can storedfor a period of at least about 52 weeks or at least 24 months at atemperature of between 2 to 8° C., preferably 5° C. wherein there isless than 20% increase in aggregation of the antibody of thecomposition. Preferably the concentration of antibody is any of about2.5, 5, 10, 20 or 50 mg/ml.

Preferably there is less than about 15%, further preferably less thanany of about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5% orequal to 0% increase in aggregation of the antibody of the liquidformulation when stored for a period of any of at least about 2, 4, 8,13, 26 or 52 weeks, further preferably for any of at least about 2, 3,6, 9, 12, 18 or 24 months at a temperature of about 2 to 8° C.,preferably 5° C. in comparison to the same composition, an equivalentsample of identical or about identical composition, prior to the periodof storage. Preferably the concentration of antibody is any of about2.5, 5, 10, 20 or 50 mg/ml.

According to a further aspect of the present invention there is provideda liquid composition of the invention wherein the composition can storedfor a period of at least about 52 weeks or at least 24 months at atemperature of between 2 to 8° C., preferably 5° C. wherein there isless than 20% increase in oxidation of the antibody of the composition.Preferably the concentration of antibody is any of about 2.5, 5, 10, 20or 50 mg/ml.

Preferably there is less than about 15%, further preferably less thanany of about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5% orequal to 0% increase in oxidation of the antibody of the liquidformulation when stored for a period of any of at least about 2, 4, 8,13, 26 or 52 weeks, further preferably for any of at least about 2, 3,6, 9, 12, 18 or 24 months at a temperature of about 2 to 8° C.,preferably 5° C. in comparison to the same composition, an equivalentsample of identical or about identical composition, prior to the periodof storage. Preferably the concentration of antibody is any of about2.5, 5, 10, 20 or 50 mg/ml.

According to a further aspect of the present invention there is provideda liquid composition of the invention wherein the composition can storedfor a period of at least about 52 weeks or at least 24 months at atemperature of between 2 to 8° C., preferably 5° C. wherein there isless than 20% increase in fragmentation of the antibody of thecomposition. Preferably the concentration of antibody is any of about2.5, 5, 10, 20 or 50 mg/ml.

Preferably there is less than about 15%, further preferably less thanany of about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5% orequal to 0% increase in fragmentation of the antibody of the liquidformulation when stored for a period of any of at least about 2, 4, 8,13, 26 or 52 weeks, further preferably for any of at least about 2, 3,6, 9, 12, 18 or 24 months at a temperature of about 2 to 8° C.,preferably 5° C. in comparison to the same composition, an equivalentsample of identical or about identical composition, prior to the periodof storage. Preferably the concentration of antibody is any of about2.5, 5, 10, 20 or 50 mg/ml.

According to a further preferred aspect of the present invention thereis provided a liquid composition, according to any foregoing aspect orembodiment, for the manufacture of a medicament for treatment of pain ina mammal.

Preferably the pain is selected from one or more of acute pain, chronicpain, neuropathic, inflammatory, nociceptive, mixed aetiology pain,hyperalgesia, allodynia, visceral pain, somatic pain and back pain.

According to a yet further embodiment of the invention there is provideda liquid composition, according to any foregoing aspect or embodiment,for the manufacture of a medicament for treatment of pain and/or lowerurinary tract symptoms (LUTs) associated with interstitial cystitisand/or painful bladder syndrome and/or bladder pain syndrome,

According to a yet further embodiment of the invention there is provideda liquid composition, according to any foregoing aspect or embodiment,for the manufacture of a medicament for treatment of pain and/or chronicprostatitis and/or chronic pelvic pain syndrome. According to anotheraspect there is provided a liquid composition, according to anyforegoing aspect or embodiment, for the manufacture of a medicament fortreatment of pain and/or other symptoms associated with endometriosisand or uterine fibroids.

Preferably the mammal is selected from rodents (such as mice, rats andrabbits, pets (such as cats, dogs and horses), farm animals (such ascows, sheep, pigs and goats), sport animals and/or pets (such as cats,dogs and horses), primates, more preferably a human.

According to a preferred embodiment the liquid composition can beadministered directly into the blood stream, into muscle, into tissue,into fat, or into an internal organ. Suitable means for parenteraladministration include intravenous, intraarterial, intraperitoneal,intrathecal, intraventricular, intraurethral, intrasternal,intracranial, intramuscular, intra-ossial, intradermal and subcutaneous.Suitable devices for parenteral administration include needle (includingmicroneedle, microprojections, soluble needles and other microporeformation techniques) injectors, needle-free injectors and infusiontechniques.

In some embodiments the administration pattern of the medicamentcomprises administration of a dose of the medicament once every week,once every two weeks, once every three weeks, once every four weeks,once every five weeks, once every six weeks, once every seven weeks,once every eight weeks, once every nine weeks, once every ten weeks,once every fifteen weeks, once every twenty weeks, once every twentyfive weeks, or once every twenty six weeks. In some embodiments, theanti-NGF antagonist antibody is administered once every month, onceevery two months, once every three months, once every four months, onceevery five months, or once every six months. Most preferably theadministration pattern of the medicament comprises administration of adose of the medicament once every eight weeks.

In some embodiments the volume of a dose is less than or equal to about20 ml, about 15 ml, about 10 ml, about 5 ml, about 2.5 ml, about 1.5 ml,about 1.0 ml, about 0.75 ml, about 0.5 ml, about 0.25 ml or about 0.01ml.

In some embodiments the volume of a dose is about 20 ml, about 19 ml,about 18 ml, about 17 ml, about 16 ml, about 15 ml, about 14 ml, about13 ml, about 12 ml, about 11 ml, about 10 ml, about 9 ml, about 8 ml,about 7 ml, about 6 ml, about 5 ml, about 4 ml, about 3 ml, about 2 mlor about 1 ml. Alternatively about 20.5 ml, about 19.5 ml, about 18.5ml, about 17.5 ml, about 16.5 ml, about 15.5 ml, about 14.5 ml, about13.5 ml, about 12.5 ml, about 11.5 ml, about 10.5 ml, about 9.5 ml,about 8.5 ml, about 7.5 ml, about 6.5 ml, about 5.5 ml, about 4.5 ml,about 3.5 ml, about 2.5 ml, about 1.5 ml, or about 0.5. Alternativelyabout 900 microliters, about 800 microliters, about 700 microliters,about 600 microliters, about 500 microliters, about 400 microliters,about 300 microliters, about 200 microliters, or about 100 microliters,alternatively about 950 microliters, about 850 microliters, about 750microliters, about 650 microliters, about 550 microliters, about 450microliters, about 350 microliters, about 250 microliters, about 150microliters, or about 50 microliters. Most preferably the volume of thedose is less than or equal to about 2.5 ml.

According to preferred embodiment the concentration of antibody canrange from about 0.1 to about 200 mg/ml. Preferably the concentration ofantibody is about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 2.5mg/ml, about 3 mg/ml, about 3.5 mg/ml, about 4 mg/ml, about 4.5 mg/ml,about 5 mg/ml, about 5.5 mg/ml, about 6 mg/ml, about 6.5 mg/ml, about 7mg/ml, about 7.5 mg/ml, about 8 mg/ml, about 8.5 mg/ml, about 9 mg/ml,about 9.5 mg/ml, about 10 mg/ml, about 11 mg/ml, about 12 mg/ml, about13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml, about 21 mg/ml,about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25 mg/ml, about 26mg/ml, about 27 mg/ml, about 28 mg/ml, about 29 mg/ml, about 30 mg/ml,about 31 mg/ml, about 32 mg/ml, about 33 mg/ml, about 34 mg/ml, about 35mg/ml, about 36 mg/ml, about 37 mg/ml, about 38 mg/ml, about 39 mg/ml,about 40 mg/ml, about 41 mg/ml, about 42 mg/ml, about 43 mg/ml, about 44mg/ml, about 45 mg/ml, about 46 mg/ml, about 47 mg/ml, about 48 mg/ml,about 49 mg/ml, about 50 mg/ml, about 51 mg/ml, about 52 mg/ml, about 53mg/ml, about 54 mg/ml, about 55 mg/ml, about 56 mg/ml, about 57 mg/ml,about 58 mg/ml, about 59 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80mg/ml, about 90 mg/ml, about 100 mg/ml or about 110 mg/ml. Mostpreferably the concentration of antibody is less than or equal to 50mg/ml and may be selected from the group comprising about 2 mg/ml, about2.5 mg/ml, about 5 mg/ml, about 10 mg/ml, about 19 mg/ml, about 20mg/ml, about 22 mg·ml and about 50 mg/ml.

According to a preferred embodiment the dose contains less than or equalto about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg,about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg,about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg,about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg,about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg,about 70 mg, about 80 mg, about 90 mg, about 100 mg, or about 110 mg ofantibody. Most preferable the dose contains less than or equal to about50 mg of antibody.

According to a preferred embodiment the dose contains an amount ofantibody that is about 1 μg/kg, about 10 μg/kg, about 20 μg/kg, about 25μg/kg, about 50 μg/kg, about 100 μg/kg, about 200 μg/kg, about 250μg/kg, about 500 μg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg,about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8mg/kg, about 9 mg/kg, about 10 mg/kg, or about 11 mg/kg (of mass of themammal to which the dose it to be administered). Most preferably thedose contains about 20 μg/kg, about 25 μg/kg, about 50 μg/kg, about 100μg/kg, about 200 μg/kg, about 250 μg/kg, 1 mg/kg or about 2 mg/kg.

Dosage regimens may depend on the pattern of pharmacokinetic decay thatthe practitioner wishes to achieve. For example, in some embodiments,dosing from one-four times a week is contemplated. Even less frequentdosing may be used. In some embodiments, the dose is administered onceevery 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks,every 15 weeks, every 20 weeks, every 25 weeks, or longer. In someembodiments, the dose is administered once every 1 month, every 2months, every 3 months, every 4 months, every 5 months, every 6 months,or longer. Most preferably the dose is administered once every eightweeks. The progress of this therapy is easily monitored by conventionaltechniques and assays. The dosing regimen can vary over time.

For the purpose of the present invention, the appropriate dosage of themedicament will depend on the antibody employed, the type and severityof the pain to be treated, whether the agent is administered forpreventative or therapeutic purposes, previous therapy, the patient'sclinical history and response to the agent, and the discretion of theattending physician. Typically the clinician will administer themedicament, until a dosage is reached that achieves the desired result.Dosages may be determined empirically for example individuals are givenincremental dosages to assess efficacy of the medicament, an indicatorof pain may be followed, such as a change in a pain numerical ratingscale (NRS).

Dose and/or frequency can vary over course of treatment. Empiricalconsiderations, such as the antibody half-life, generally willcontribute to the determination of the dosage. Frequency ofadministration may be determined and adjusted over the course oftherapy, and is generally, but not necessarily, based on treatmentand/or suppression and/or amelioration and/or delay of pain. In someindividuals, more than one dose may be required. Frequency ofadministration may be determined and adjusted over the course oftherapy. For repeated administrations over several days or longer,depending on the pain and its severity, the treatment is sustained untila desired suppression of symptoms occurs or until sufficient therapeuticlevels are achieved to reduce pain.

Administration of medicament comprising the liquid composition can becontinuous or intermittent, depending, for example, upon the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of the medicament comprising theliquid composition may be essentially continuous over a preselectedperiod of time or may be in a series of spaced dose, e.g., eitherbefore, during, or after developing pain.

Preferably the administration of the dose is a parenteral administrationpreferably selected from intravenous, intraarterial, intraperitoneal,intrathecal, intraventricular, intraurethral, intrasternal,intracranial, intramuscular, intra-ossial, intradermal and subcutaneous.Preferably the medicament is in a unit dosage sterile liquid form forparenteral administration.

Treatment efficacy can be assessed by monitoring pain relief. Painrelief may be characterized by time course of relief. Accordingly, insome embodiments, relief is observed within about 24 hours afteradministration. In other embodiments, relief is observed within about36, 48, 60, 72 hours or 4 days after administration. In someembodiments, frequency and/or intensity of pain is diminished, and/orquality of life of those suffering pain is increased. In someembodiments, pain relief is provided for duration of at least about 7days, at least about 14 days, at least about 21 days, at least about 28days, at least about 35 days, at least about 42 days, at least about 49days, at least about 56 days, at least about 63 days, at least about 70days, at least about 77 days, at least about 84 days, at least about 180days, or longer after a single dose of the medicament.

Combinations

The liquid composition, according to any aspect or embodiment may beusefully combined with another pharmacologically active compound, orwith two or more other pharmacologically active compounds, particularlyin the treatment of pain in a mammal and may be administeredsimultaneously, sequentially or separately in combination with one ormore agents selected from:

-   (i) an opioid analgesic, e.g. morphine, heroin, hydromorphone,    oxymorphone, levorphanol, levallorphan, methadone, meperidine,    fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone,    propoxyphene, nalmefene, nalorphine, naloxone, naltrexone,    buprenorphine, butorphanol, nalbuphine or pentazocine;-   (ii) a nonsteroidal antiinflammatory drug (NSAID), e.g. aspirin,    diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal,    flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac,    meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin,    phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, or a    pharmaceutically acceptable salt thereof;-   (iii) a barbiturate sedative, e.g. amobarbital, aprobarbital,    butabarbital, butabital, mephobarbital, metharbital, methohexital,    pentobarbital, phenobartital, secobarbital, talbutal, theamylal or    thiopental or a pharmaceutically acceptable salt thereof;-   (iv) a benzodiazepine having a sedative action, e.g.    chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam,    oxazepam, temazepam or triazolam or a pharmaceutically acceptable    salt thereof,-   (v) an H₁ antagonist having a sedative action, e.g. diphenhydramine,    pyrilamine, promethazine, chlorpheniramine or chlorcyclizine or a    pharmaceutically acceptable salt thereof;-   (vi) a sedative such as glutethimide, meprobamate, methaqualone or    dichloralphenazone or a pharmaceutically acceptable salt thereof;-   (vii) a skeletal muscle relaxant, e.g. baclofen, carisoprodol,    chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenadine or a    pharmaceutically acceptable salt thereof,-   (viii) an NMDA receptor antagonist, e.g. dextromethorphan    ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan    ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine,    pyrroloquinoline quinone or    cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid or a    pharmaceutically acceptable salt thereof;-   (ix) an alpha-adrenergic, e.g. doxazosin, tamsulosin, clonidine or    4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl)    quinazoline;-   (x) a tricyclic antidepressant, e.g. desipramine, imipramine,    amytriptiline or nortriptiline;-   (xi) an anticonvulsant, e.g. carbamazepine or valproate;-   (xii) a tachykinin (NK) antagonist, particularly an NK-3, NK-2 or    NK-1 antagonist, e.g.    (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]naphthridine-6-13-dione    (TAK-637),    5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one    (MK-869), lanepitant, dapitant or    3-[[2-methoxy-5-(trifluoromethoxy)phenyl]methylamino]-2-phenyl-piperidine    (2S,3S);-   (xiii) a muscarinic antagonist, e.g oxybutin, tolterodine,    propiverine, tropsium chloride or darifenacin;-   (xiv) a COX-2 inhibitor, e.g. celecoxib, rofecoxib or valdecoxib;-   (xv) a non-selective COX inhibitor (preferably with GI protection),    e.g. nitroflurbiprofen (HCT-1026);-   (xvi) a coal-tar analgesic, in particular paracetamol;-   (xvii) a neuroleptic such as droperidol;-   (xviii) a vanilloid receptor agonist (e.g. resinferatoxin) or    antagonist (e.g. capsazepine);-   (xix) a beta-adrenergic such as propranolol;-   (xx) a local anaesthetic, such as mexiletine;-   (xxi) a corticosteriod, such as dexamethasone-   (xxii) a serotonin receptor agonist or antagonist;-   (xxiii) a cholinergic (nicotinic) analgesic;-   (xxiv) Tramadol (trade mark);-   (xxv) a PDEV inhibitor, such as sildenafil, vardenafil or taladafil;-   (xxvi) an alpha-2-delta ligand such as gabapentin or pregabalin; and-   (xxvii) a canabinoid.

DESCRIPTION OF THE FIGURES

FIG. 1 Summary of % Aggregation (via SEC) Data from 50 mg/mL anti-NGFantibody: Tonicity Agent Screening Study at 40° C.

FIG. 2. Summary of % Fragments (via reduced CGE) Data from 50 mg/mLanti-NGF antibody: Tonicity Agent Screening Study at 40° C.

FIG. 3 Summary of % Aggregation (via SEC) Data from 22 mg/mL anti-NGFantibody subjected to 15 Freeze-Thaw Cycles:

FIG. 4 % Aggregation Data (per SEC) at 5° C. for Long-Term StabilityStudy of anti-NGF antibody E3 formulation containing trehalose, EDTA,polysorbate 20 in histidine buffer at pH 6.0 at 2.5, 5, 10, 20 and 50mg/mL

FIG. 5 % Fragmentation Data (per reduced CGE) at 5° C. for Long-TermStability Study of anti-NGF antibody E3 formulation containingtrehalose, EDTA, polysorbate 20 in histidine buffer at pH 6.0 at 2.5, 5,10, 20 and 50 mg/mL

FIG. 6 % Oxidation Data at 5° C. for Long-Term Stability Study ofanti-NGF antibody E3 formulation containing trehalose, EDTA, polysorbate20 in histidine buffer at pH 6.0 at 2.5, 5, 10, 20 and 50 mg/mL

DEFINITIONS

As used herein, the terms “formulation” or “composition” as they relateto an antibody are meant to describe the antibody in combination with apharmaceutically acceptable excipient comprising at least one tonicityagent, at least one buffer, at least one chelating agent, at least onesurfactant, wherein the pH is as defined.

The term “pharmaceutical composition” refers to preparations which arein such form as to permit the biological activity of the activeingredients to be effective.

“Pharmaceutically acceptable excipients” (vehicles, additives) arethose, which can safely be administered to a subject to provide aneffective dose of the active ingredient employed. The term “excipient”or “carrier” as used herein refers to an inert substance, which iscommonly used as a diluent, vehicle, preservative, binder or stabilizingagent for drugs. As used herein, the term “diluent” refers to apharmaceutically acceptable (safe and non-toxic for administration to ahuman) solvent and is useful for the preparation of the liquidformulations herein. Exemplary diluents include, but are not limited to,sterile water and bacteriostatic water for injection (BWFI).

As used herein, the term “antibody” refers to an intact antibody or anantigen-binding portion that competes with the intact antibody forspecific binding. See generally, Fundamental Immunology, Ch. 7 (Paul,W., ed., 2nd ed. Raven Press, N.Y. (1989). Antigen-binding portions maybe produced by recombinant DNA techniques or by enzymatic or chemicalcleavage of intact antibodies. In some embodiments, antigen-bindingportions include Fab, Fab′, F(ab′)2, Fd, Fv, dAb, and complementaritydetermining region (CDR) fragments, single-chain antibodies (scFv),chimeric antibodies, diabodies and polypeptides that contain at least aportion of an antibody that is sufficient to confer specific antigenbinding to the polypeptide. From N-terminus to C-terminus, both themature light and heavy chain variable domains comprise the regions FR1,CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids toeach domain is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol.196:901-917 (1987), or Chothia et al., Nature 342:878-883 (1989). Asused herein, the term “polypeptide” encompasses native or artificialproteins, protein fragments and polypeptide analogs of a proteinsequence. A polypeptide may be monomeric or polymeric.

As used herein, an Fd fragment means an antibody fragment that consistsof the V_(H) and CH1 domains; an Fv fragment consists of the V₁ andV_(H) domains of a single arm of an antibody; and a dAb fragment (Wardet al., Nature 341:544-546 (1989)) consists of a V_(H) domain.

The term “or an antigen-binding portion thereof” when used with the term“antibody” refers to a polypeptide that has an amino-terminal and/orcarboxy-terminal deletion, but where the remaining amino acid sequenceis identical to the corresponding positions in the naturally-occurringsequence. In some embodiments, fragments are at least 5, 6, 8 or 10amino acids long. In other embodiments, the fragments are at least 14,at least 20, at least 50, or at least 70, 80, 90, 100, 150 or 200 aminoacids long.

As used herein, the term “monoclonal antibody” refers to a homogeneousantibody population wherein the monoclonal antibody is comprised ofamino acids (naturally occurring and non-naturally occurring) that areinvolved in the selective binding of an antigen. A population ofmonoclonal antibodies is highly specific, being directed against asingle antigenic site. The term “monoclonal antibody” encompasses notonly intact monoclonal antibodies and full-length monoclonal antibodies,but also fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), singlechain (ScFv), mutants thereof, fusion proteins comprising an antibodyportion, and any other modified configuration of the immunoglobulinmolecule that comprises an antigen recognition site of the requiredspecificity and the ability to bind to an antigen. It is not intended tobe limited as regards to the source of the antibody or the manner inwhich it is made (e.g., by hybridoma, phage selection, recombinantexpression, transgenic animals, etc.).

A single chain antibody (scFc) is an antibody in which V_(L) and V_(H)regions are paired to form a monovalent molecule via a synthetic linkerthat enables them to be made as a single protein chain (Bird et alScience, 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci.USA, 85:5879-5883 (1988)).

Diabodies are bivalent, bispecific antibodies in which V_(H) and V_(L)domains are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites.

“Chimeric antibodies” refers to those antibodies wherein one portion ofeach of the amino acid sequences of heavy and light chains is homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular class, while the remaining segmentof the chains is homologous to corresponding sequences in another.Typically, in these chimeric antibodies, the variable region of bothlight and heavy chains mimics the variable regions of antibodies derivedfrom one species of mammals, while the constant portions are homologousto the sequences in antibodies derived from another. One clear advantageto such chimeric forms is that, for example, the variable regions canconveniently be derived from presently known sources using readilyavailable hybridomas or B cells from non human host organisms incombination with constant regions derived from, for example, human cellpreparations. While the variable region has the advantage of ease ofpreparation, and the specificity is not affected by its source, theconstant region being human, is less likely to elicit an immune responsefrom a human subject when the antibodies are injected than would theconstant region from a non-human source. However, the definition is notlimited to this particular example.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Chothia et al. (1989) Nature342:877; Al-Iazikani et al (1997) J. Molec. Biol. 273:927-948)). As usedherein, a CDR may refer to CDRs defined by either approach or by acombination of both approaches.

A “constant region” of an antibody refers to the constant region of theantibody light chain or the constant region of the antibody heavy chain,either alone or in combination.

As used herein, the terms “E3”, “3E”, and “antibody E3” are usedinterchangeably to refer to an antibody comprising the amino acidsequence of the heavy chain and light chain variable regions shown inSEQ ID NO:1 and SEQ ID NO:2 (FIGS. 1A and 1B of WO2004/058184),respectively. The generation and characterization of E3 is described inthe Examples of WO2004/058184, the entire content of which is hereinincorporated by reference. In some embodiments, the term “E3” refers toimmunoglobulin encoded by (a) a polynucleotide encoding E3 light chainthat has a deposit number of ATCC No. PTA-4893 or ATCC No. PTA-4894, and(b) a polynucleotide encoding E3 heavy chain that has a deposit numberof ATCC No. PTA-4895.

As used herein, the terms “isolated antibody” or “purified antibody”refers to an antibody that by virtue of its origin or source ofderivation has one to four of the following: (1) is not associated withnaturally associated components that accompany it in its native state,(2) is free of other proteins from the same species, (3) is expressed bya cell from a different species, or (4) does not occur in nature.

An antibody is “substantially pure,” “substantially homogeneous,” or“substantially purified” when at least about 60 to 75% of a sampleexhibits a single species of antibody. A substantially pure antibody cantypically comprise about 50%, 60%, 70%, 80% or 90% w/w of an antibodysample, more usually about 95%, and preferably will be over 99% pure.Antibody purity or homogeneity may tested by a number of means wellknown in the art, such as polyacrylamide gel electrophoresis or HPLC.

As used herein, the term “human antibody” is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. This definition of a human antibodyincludes antibodies comprising at least one human heavy chainpolypeptide or at least one human light chain polypeptide. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

As used herein, “humanized” antibody refers to forms of non-human (e.g.murine) antibodies that are chimeric immunoglobulins, immunoglobulinchains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen-binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. Preferably, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat, or rabbit having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, the humanized antibody may compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences, but are included to further refineand optimize antibody performance. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region or domain (Fc),typically that of a human immunoglobulin. Preferred are antibodieshaving Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDRH1, CDR H2, or CDR H3) which are altered with respect to the originalantibody, which are also termed one or more CDRs “derived from” one ormore CDRs from the original antibody.

There are four general steps to humanize a monoclonal antibody. Theseare: (1) determining the nucleotide and predicted amino acid sequence ofthe starting antibody light and heavy variable domains (2) designing thehumanized antibody, i. e., deciding which antibody framework region touse during the humanizing process (3) the actual humanizingmethodologies/techniques and (4) the transfection and expression of thehumanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;5,585,089; and 6,180,370.

A number of “humanized” antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have beendescribed, including chimeric antibodies having rodent or modifiedrodent V regions and their associated complementarity determiningregions (CDRs) fused to human constant domains. See, for example, Winteret al. Nature 349: 293-299 (1991), Lobuglio et al. Proc. Nat. Acad. Sci.USA 86: 4220-4224 (1989), Shaw et al. J Immunol. 138: 4534-4538 (1987),and Brown et al. Cancer Res. 47: 3577-3583 (1987). Other referencesdescribe rodent CDRs grafted into a human supporting framework region(FR) prior to fusion with an appropriate human antibody constant domain.See, for example, Riechmann et al. Nature 332: 323-327 (1988), Verhoeyenet al. Science 239: 1534-1536 (1988), and Jones et al. Nature 321:522-525 (1986). Another reference describes rodent CDRs supported byrecombinantly veneered rodent framework regions. See, for example,European Patent Publication No. 0519596.

These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent anti-human antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients. For example, the antibody constantregion can be engineered such that it is immunologically inert (e. g.,does not trigger complement lysis). See, e. g. PCT Publication No.WO99/58572; UK Patent Application No. 9809951.8. Other methods ofhumanizing antibodies that may also be utilized are disclosed byDaugherty et al., Nucl. Acids Res. 19: 2471-2476 (1991) and in U.S. Pat.Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and6,350,861; and in PCT Publication No. WO 01/27160.

As used herein, the term “recombinant antibody” is intended to includeall antibodies that are prepared, expressed, created or isolated byrecombinant means, for example antibodies expressed using a recombinantexpression vector transfected into a host cell, antibodies isolated froma recombinant, combinatorial human antibody library, antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes or antibodies prepared, such recombinant humanantibodies can be subjected to in vitro mutagenesis.

As used herein, the terms “is capable of specifically binding”,“specifically binds”, or “preferentially binds” refers to when anantibody binds to an antigen with a dissociation constant that is ≤1 μM,preferably ≤1 nM and most preferably ≤10 pM.

An epitope that “specifically binds”, or “preferentially binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody“specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an NGF epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other NGF epitopes or non-NGFepitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding.

As used herein, “immunospecific” binding of antibodies refers to theantigen specific binding interaction that occurs between theantigen-combining site of an antibody and the specific antigenrecognized by that antibody (i.e., the antibody reacts with the proteinin an ELISA or other immunoassay, and does not react detectably withunrelated proteins).

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen-binding portion thereof, binds toan epitope in a manner sufficiently similar to the binding of a secondantibody, or an antigen-binding portion thereof, such that the result ofbinding of the first antibody with its cognate epitope is detectablydecreased in the presence of the second antibody compared to the bindingof the first antibody in the absence of the second antibody. Thealternative, where the binding of the second antibody to its epitope isalso detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its cognate epitope or ligand, whether to the same,greater, or lesser extent, the antibodies are said to “cross-compete”with each other for binding of their respective epitope(s). Bothcompeting and cross-competing antibodies are encompassed by the presentinvention. Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof), the skilled artisanwould appreciate, based upon the teachings provided herein, that suchcompeting and/or cross-competing antibodies are encompassed and can beuseful for the methods disclosed herein.

As used herein, the term “nerve growth factor” and “NGF” refers to nervegrowth factor and variants thereof that retain at least part of thebiological activity of NGF. As used herein, NGF includes all mammalianspecies of native sequence NGF, including human, canine, feline, equine,or bovine.

“NGF receptor” refers to a polypeptide that is bound by or activated byNGF. NGF receptors include the TrkA receptor and the p75 receptor of anymammalian species, including, but are not limited to, human, canine,feline, equine, primate, or bovine.

As used herein, an “anti-NGF antibody” (interchangeably termed “anti-NGFantagonist antibody”) refers to an antibody which is able to bind to NGFand inhibit, block, antagonize, suppress or reduce NGF biologicalactivity and/or downstream pathway(s) mediated by NGF signaling. In someembodiments the term “anti-NGF antagonist antibody” encompass all thepreviously identified terms, titles, and functional states andcharacteristics whereby the NGF itself, an NGF biological activity(including but not limited to its ability to ability to mediate anyaspect of post-surgical pain), or the consequences of the biologicalactivity, are substantially nullified, decreased, or neutralized in anymeaningful degree. In some embodiments, an anti-NGF antagonist antibodybinds NGF and prevent NGF dimerization and/or binding to an NGF receptor(such as p75 and/or trkA). In other embodiments, an anti-NGF antibodybinds NGF and prevents trkA receptor dimerization and/or trkAautophosphorylation. Examples of anti-NGF antagonist antibodies areprovided herein.

The term “identity” refers to the percent “identity” of two amino acidsequences or of two nucleic acid sequences. The percent identity isgenerally determined by aligning the sequences for optimal comparisonpurposes (e.g. gaps can be introduced in the first sequence for bestalignment with the second sequence) and comparing the amino acidresidues or nucleotides at corresponding positions. The “best alignment”is an alignment of two sequences that results in the highest percentidentity. The percent identity is determined by comparing the number ofidentical amino acid residues or nucleotides within the sequences (i.e.,% identity=number of identical positions/total number of positions×100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programsof Altschul, et al (1990) J. Mol. Biol. 215:403-410 have incorporatedsuch an algorithm. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucliec Acids Res. 25:3389-3402.

Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules (Id.) When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). The ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package has incorporated such analgorithm. Other algorithms for sequence analysis known in the artinclude ADVANCE and ADAM as described in Torellis and Robotti (1994)Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a controloption that sets the sensitivity and speed of the search.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, which in the context of anti-NGF antibodies includestreatment or prophylactic prevention of the targeted pathologiccondition for example inflammation or pain. It is to be noted thatdosage values may vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.Likewise, a therapeutically effective amount of the antibody or antibodyportion may vary according to factors such as the disease state, age,sex, and weight of the individual, the ability of the antibody orantibody portion to elicit a desired response in the individual, and thedesired route of administration of the antibody formulation. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects.

As used herein, the term “treatment” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic conditionfor example pain. Those in need of treatment include those already withthe condition as well as those prone to have the condition or those inwhom the condition is to be prevented. As used herein, “treatment” is anapproach for obtaining beneficial or desired clinical results including,but not limited to, one or more of the following: including lesseningseverity, alleviation of one or more symptoms associated with pain,including any aspect of pain, (such as shortening duration of pain,reduction of pain sensitivity or sensation).

An “effective amount” of drug, compound, or pharmaceutical compositionis an amount sufficient to effect beneficial or desired resultsincluding clinical results such as alleviation or reduction of thetargeted pathologic condition for example pain sensation. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of drug, compound, orpharmaceutical composition is an amount sufficient to treat, ameliorate,reduce the intensity of the targeted pathologic condition for exampleinflammation or pain. In some embodiments, the “effective amount” mayreduce pain at rest (resting pain) or mechanically-induced pain(including pain following movement), or both, and it may be administeredbefore, during or after painful stimulus. As is understood in theclinical context, an effective amount of a drug, compound, orpharmaceutical composition may or may not be achieved in conjunctionwith another drug, compound, or pharmaceutical composition. Thus, an“effective amount” may be considered in the context of administering oneor more therapeutic agents, and a single agent may be considered to begiven in an effective amount if, in conjunction with one or more otheragents, a desirable result may be or is achieved.

“Pain” as used herein refers to pain of any etiology, including acuteand chronic pain, and any pain with an inflammatory component. As usedherein, “pain” includes nociception and the sensation of pain, and paincan be assessed objectively and subjectively, using pain scores andother methods well-known in the art. The pain can be primary orsecondary pain, as is well-known in the art.

As used herein, the term “subject” for purposes of treatment includesany subject, and preferably is a subject who is in need of the treatmentof the targeted pathologic condition for example inflammation or pain.For purposes of prevention, the subject is any subject, and preferablyis a subject that is at risk for, or is predisposed to, developing thetargeted pathologic condition for example inflammation or pain. The term“subject” is intended to include living organisms, e.g., prokaryotes andeukaryotes. Examples of subjects include mammals, e.g., humans, dogs,cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, andtransgenic non-human animals. In specific embodiments of the invention,the subject is a human.

When introducing elements of the present invention or the preferredembodiment(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “comprise”, “comprises”, “including” and “having” areintended to be inclusive and mean that there may be additional elementsother than the listed elements.

As used herein, the term “polynucleotide” or “nucleic acid”, usedinterchangeably herein, means a polymeric form of nucleotides eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide and may be single and double stranded forms. A“polynucleotide” or a “nucleic acid” sequence encompasses its complementunless otherwise specified. As used herein, the term “isolatedpolynucleotide” or “isolated nucleic acid” means a polynucleotide ofgenomic, cDNA, or synthetic origin or some combination thereof, which byvirtue of its origin or source of derivation, the isolatedpolynucleotide has one to three of the following: (1) is not associatedwith all or a portion of a polynucleotide with which the “isolatedpolynucleotide” is found in nature, (2) is operably linked to apolynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

As used herein, the term “chelating agent” is an excipient that can format least one bond (e.g., covalent, ionic, or otherwise) to a metal ion.A chelating agent is typically a multidentate ligand that can be used inliquid compositions as a stabilizer to complex with species, which mightotherwise promote instability.

As used herein, the term “buffer” refers to an added composition thatallows a liquid antibody formulation to resist changes in pH, typicallyby action of its acid-base conjugate components. When a concentration ofa buffer is referred to, it is intended that the recited concentrationrepresent the molar concentration of the free acid or free base form ofthe buffer.

As used herein, the terms ‘tonicity agent” or “tonicifier” refers to anexcipient that can adjust the osmotic pressure of a liquid antibodyformulation. In certain embodiments, the tonicity agent can adjust theosmotic pressure of a liquid antibody formulation to isotonic so thatthe antibody formulation is physiologically compatible with the cells ofthe body tissue of the subject. In still other embodiments, the“tonicity agent” may contribute to an improvement in stability ofantibodies described herein. An “isotonic” formulation is one that hasessentially the same osmotic pressure as human blood. Isotonicformulations generally have an osmotic pressure from about 250 to 350mOsm. The term “hypotonic” describes a formulation with an osmoticpressure below that of human blood. Correspondingly, the term“hypertonic” is used to describe a formulation with an osmotic pressureabove that of human blood, Isotonicity can be measured using a vaporpressure or ice-freezing type osmometer, for example. The tonicity agentcan be in an enantiomeric (e.g., L- or D-enantiomer) or racemic form;isomers such as alpha or beta, including alpha, alpha; or beta, beta; oralpha, beta; or beta, alpha; a free acid or free base form; a hydratedform (e.g., monohydrate), or an anhydrous form.

As used herein, the term “polyol” refers an excipient with multiplehydroxyl groups, and includes sugars (reducing and nonreducing sugars),sugar alcohols and sugar acids.

As used herein, the term “surfactant” refers to an excipient that canalter the surface tension of a liquid antibody formulation. In certainembodiments, the surfactant reduces the surface tension of a liquidantibody formulation. In still other embodiments, the “surfactant” maycontribute to an improvement in stability of any of the antibody in theformulation. The surfactant may reduce aggregation of the formulatedantibody and/or minimize the formation of particulates in theformulation and/or reduces adsorption. The surfactant may also improvestability of the antibody during and after a freeze/thaw cycle.

As used herein, the term “saccharide” refers to a class of moleculesthat are derivatives of polyhydric alcohols. Saccharides are commonlyreferred to as carbohydrates and may contain different amounts of sugar(saccharide) units, e.g., monosaccharides, disaccharides andpolysaccharides.

As used herein, the term “reducing sugar” is one which contains ahemiacetal group that can reduce metal ions or react covalently withlysine and other amino groups in proteins and a “nonreducing sugar” isone which does not have these properties of a reducing sugar.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with an active ingredient, allows theingredient to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline. Compositions comprising such carriers are formulated by wellknown conventional methods (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton,Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing, 2000).

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “Kd”, as used herein, is intended to refer to the dissociationconstant of an antibody-antigen interaction. One way of determining theKd or binding affinity of antibodies to NGF is by measuring bindingaffinity of monofunctional Fab fragments of the antibody. To obtainmonofunctional Fab fragments, an antibody (for example, IgG) can becleaved with papain or expressed recombinantly. The affinity of ananti-NGF Fab fragment of an antibody can be determined by surfaceplasmon resonance (BIAcore3000™ surface plasmon resonance (SPR) system,BIAcore, INC, Piscaway N.J.). CM5 chips can be activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Human NGF (or any other NGF) can be diluted into 10 mM sodium acetate pH4.0 and injected over the activated chip at a concentration of 0.005mg/mL. Using variable flow time across the individual chip channels, tworanges of antigen density can be achieved: 100-200 response units (RU)for detailed kinetic studies and 500-600 RU for screening assays. Serialdilutions (0.1-10× estimated K_(D)) of purified Fab samples are injectedfor 1 min at 100 microliters/min and dissociation times of up to 2 h areallowed. The concentrations of the Fab proteins are determined by ELISAand/or SDS-PAGE electrophoresis using a Fab of known concentration (asdetermined by amino acid analysis) as a standard. Kinetic associationrates (k_(on)) and dissociation rates (k_(off)) are obtainedsimultaneously by fitting the data to a 1:1 Langmuir binding model(Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values are calculated as k_(off)/k_(on).This protocol is suitable for use in determining binding affinity of anantibody to any NGF, including human NGF, NGF of another vertebrate (insome embodiments, mammalian) (such as mouse NGF, rat NGF, primate NGF),as well as for use with other neurotrophins, such as the relatedneurotrophins NT3, NT4/5, and/or BDNF.

Anti-NGF antagonist antibodies for use in the invention can beidentified or characterized using methods known in the art, wherebyreduction, amelioration, or neutralization of an NGF biological activityis detected and/or measured. Methods described in PCT WO 04/065560 canbe used. Another method, for example, a kinase receptor activation(KIRA) assay described in U.S. Pat. Nos. 5,766,863 and 5,891,650, can beused to identify anti-NGF agents.

Anti-NGF antagonist antibodies can also be identified by incubating acandidate agent, for example an antibody or anti-NGF antibody, with NGFand monitoring any one or more of the following characteristics: (a)binding to NGF and inhibiting NGF biological activity or downstreampathways mediated by NGF signaling function; (b) inhibiting, blocking ordecreasing NGF receptor activation (including TrkA dimerization and/orautophosphorylation); (c) increasing clearance of NGF; (d) treating orpreventing pain; (e) inhibiting (reducing) NGF synthesis, production orrelease. The ability of an anti-NGF antagonist antibody to block orneutralize a biological activity of NGF can also be assessed bymonitoring the ability of the candidate agent to inhibit NGF mediatedsurvival in the embryonic rat dorsal root ganglia survival bioassay asdescribed in Hongo et al., Hybridoma 19:215-227 (2000).

“Reducing incidence” of pain means any of reducing severity (which caninclude reducing need for and/or amount of (e. g., exposure to) otherdrugs and/or therapies generally used for this conditions, including,for example, opiates), duration, and/or frequency (including, forexample, delaying or increasing time to post-surgical pain in anindividual). As is understood by those skilled in the art, individualsmay vary in terms of their response to treatment, and, as such, forexample, a “method of reducing incidence of rheumatoid arthritis pain orosteoarthritis pain in an individual” reflects administering theanti-NGF antagonist antibody based on a reasonable expectation that suchadministration may likely cause such a reduction in incidence in thatparticular individual.

“Ameliorating” a pain or one or more symptoms of a pain (such asrheumatoid arthritis pain or osteoarthritis pain) means a lessening orimprovement of one or more symptoms of a pain as compared to notadministering an anti-NGF antagonist antibody. “Ameliorating” alsoincludes shortening or reduction in duration of a symptom.

“Palliating” a pain or one or more symptoms of a pain (such asrheumatoid arthritis pain or osteoarthritis pain) means lessening theextent of one or more undesirable clinical manifestations ofpost-surgical pain in an individual or population of individuals treatedwith an anti-NGF antagonist antibody in accordance with the invention.

As used therein, “delaying” the development of pain means to defer,hinder, slow, retard, stabilize, and/or postpone progression of pain,(such as post-surgical pain, rheumatoid arthritis pain, orosteoarthritis pain). This delay can be of varying lengths of time,depending on the history of the disease and/or individuals beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop pain. A method that “delays” development ofthe symptom is a method that reduces probability of developing thesymptom in a given time frame and/or reduces. extent of the symptoms ina given time frame, when compared to not using the method. Suchcomparisons are typically based on clinical studies, using astatistically significant number of subjects.

“Pain” is taken to mean an unpleasant sensory and emotional experienceassociated with actual or potential tissue damage or described in termsof such damage. Pain can be divided into a number of different areasbecause of differing pathophysiology, these include nociceptive,inflammatory, neuropathic pain etc. It should be noted that some typesof pain have multiple aetiologies and thus can be classified in morethan one area, e.g. Back pain, Cancer pain have both nociceptive andneuropathic components.

“Chronic pain” as used herein is taken to mean pain associated with achronic disorder, i.e. trauma, malignancy, disease, infection or painthat persists beyond resolution of such an underlying disorder, orhealing of an injury, and that is often more intense than the underlyingprocess or disorder would predict. Chronic pain may also be a “mixedaetiology pain” for example involving both nociceptive and/orneuropathic pain and/or inflammatory pain and/or cancer pain. As aconsequence chronic pain is often unpredictable in response toanalgesia. “Dual mechanism pain” is taken to mean pain that is amplifiedand maintained by both peripheral and central sensitization.

“Inflammatory pain” is taken to mean pain in response to tissue injuryand the resulting inflammatory process. Inflammatory pain may originatein injured tissue that undergoes a reactive inflammatory process whichmay also affect neuronal function. Inflammatory pain may involve thebinding of biochemical mediators (PGE2, bradykinin, cytokines, andneuropeptides), to receptors on pain-transmitting neurons and alterationof their function, increasing their excitability and thus increasingpain sensation. Included in the term inflammatory pain are chronic andacute pain.

“Mixed aetiology pain” is taken to mean pain that contains bothinflammatory and/or neuropathic and/or nociceptive components. “Dualmechanism pain” is taken to mean pain that is amplified and maintainedby both peripheral and central sensitization.

“Neuropathic pain” is taken to mean pain produced by damage to ordysfunction of neurons in the peripheral or central nervous system.

“Acute pain” and “acute inflammatory pain” is taken to mean the normal,predictable, appropriate physiological response to a noxious chemical,thermal or mechanical stimulus or to a disease process that threatens orproduces tissue injury which leads to the acute inflammation and acuteinflammatory pain response, such acute pain often involves a nociceptivepain component. In general, the intensity of the acute pain isproportional to the intensity of the stimulus and persists as long asthe stimulus persists, or until healing of tissue injury. Acuteinflammatory pain is generally associated with injury, invasiveprocedures, trauma, infection, immune reaction, allergy,hypersensitivity and disease.

“Nociceptive pain” is taken to mean pain that is transmitted bynociceptors caused by noxious stimuli signalling tissue injury orimpending tissue injury. Nociceptive pain involves transmission of painsignals via afferent neurons to the dorsal horn of the spinal cord,second order neurons transmit the signals to higher centres. Nociceptivepain normally resolves once the condition that precipitated it isresolved, also characteristic of acute pain.

“Hyperalgesia” is an extreme sensitivity to pain, often caused by damageto nociceptors in the body's soft tissues. It can be experienced infocal form is typically associated with injury, and is divided into twosubtypes: (1) Primary hyperalgesia i.e. pain sensitivity that occursdirectly in the damaged tissues, (2) secondary hyperalgesia i.e. painsensitivity that occurs in surrounding undamaged tissues. It can also beexperienced as a more diffuse, body-wide form. Hyperalgesia can beinduced by an acute or chronic inflammatory condition. Key to thedevelopment of such hyperalgesia is the action of platelet aggregatingfactor (PAF) resulting from such an inflammatory condition or from anallergic response and which occurs via immune cells interacting with theperipheral nervous system and releasing cytokines and chemokines whichlead to pain. Inflammatory conditions can induce the stimulation of painfibres in a pattern consistent with a form of amplification in thespinal cord called long term potentiation, such amplification in thespinal cord provides a pathway producing hyperalgesia.

“Allodynia” is pain resulting from stimuli which are not normallypainful, an exaggerated response to otherwise non-noxious stimuli andcan be either static or dynamic allodynia, i.e. occurring spontaneouslywithout movement or with movement. Allodynia may also be perceived inother areas than the one stimulated; hence it can also be dysesthetic.It is common to inflammatory conditions particularly joint inflammation.

“Somatic pain” is taken to mean pain originating in the cutaneous ordeep tissues, when it occurs in the musculoskeletal tissues, it iscalled “deep somatic pain”.

“Visceral pain” is taken to mean pain caused by activation of painreceptors resulting from infiltration, compression, extension, orstretching of the thoracic, abdominal, or pelvic viscera.

“Pain” as used herein refers to pain of any etiology, including acuteand chronic pain, and any pain with an inflammatory component. Examplesof pain include post-surgical pain, post-operative pain (includingdental pain), migraine, headache and trigeminal neuralgia, painassociated with burn, wound or kidney stone, pain associated with trauma(including traumatic head injury), neuropathic pain, back pain such aschronic lower back pain, pain associated with musculo-skeletal disorderssuch as rheumatoid arthritis, pain associated with osteoarthritis,ankylosing spondylitis, sero-negative (non-rheumatoid) arthropathies,non-articular rheumatism and peri-articular disorders, and painassociated with cancer (including “break-through pain”, and painassociated with terminal cancer and pain due to bone metastases),peripheral neuropathy and post-herpetic neuralgia, pain associated withinterstitial cystitis and/or painful bladder syndrome and/or bladderpain syndrome, pain associated with chronic prostatitis and/or chronicpelvic pain syndrome, pain associated with endometriosis and/or uterinefibroids. Examples of pain with an inflammatory component (in additionto some of those described above) include rheumatic pain, painassociated with mucositis, and dysmenorrhea.

“Post-surgical pain” (interchangeably termed “post-incisional” or“post-traumatic pain”) refers to pain arising or resulting from anexternal trauma such as a cut, puncture, incision, tear, or wound intotissue of an individual (including that that arises from all surgicalprocedures, whether invasive or non-invasive). As used herein,post-surgical pain does not include pain that occurs (arises ororiginates) without an external physical trauma. In some embodiments,post-surgical pain is internal or external (including peripheral) pain,and the wound, cut, trauma, tear or incision may occur accidentally (aswith a traumatic wound) or deliberately (as with a surgical incision).As used herein, “pain” includes nociception and the sensation of pain,and pain can be assessed objectively and subjectively, using pain scoresand other methods well-known in the art. Post-surgical pain, as usedherein, includes allodynia (i. e., increased response to a normallynon-noxious stimulus) and hyperalgesia (i. e., increased response to anormally noxious or unpleasant stimulus), which can in turn, be thermalor mechanical (tactile) in nature. In some embodiments, the pain ischaracterized by thermal sensitivity, mechanical sensitivity and/orresting pain. In some embodiments, the post-surgical pain comprisesmechanically-induced pain or resting pain. In other embodiments, thepost-surgical pain comprises resting pain.

The pain can be primary or secondary pain, as is well-known in the art.

Pain associated with interstitial cystitis and/or painful bladdersyndrome and/or bladder pain syndrome may comprise lower abdominal(pelvic) pain; bladder pain; suprapubic pain; vaginal pain; pain in thepenis, testicles, scrotum and perineum; urethral pain; dyspareneuria;pain, pressure or discomfort that may increase as the bladder fills.

Pain associated with chronic prostatitis and/or chronic pelvic painsyndrome may comprise lower abdominal (pelvic) pain; lower stomach pain;bladder pain; suprapubic pain; pain in the penis, testicles, scrotum andperineum; urethral pain; dyspareunia; pain, pressure or discomfort thatmay increase as the bladder fills; dysuria; and ejaculatory pain.

Lower urinary tract symptoms (LUTs) may comprise three groups of urinarysymptoms, which may be defined as storage (irritative), voiding(obstructive) and post-micturition symptoms. Storage symptoms compriseurgency, frequency, nocturia, urgency incontinence and stressincontinence, which can be associated with overactive bladder (OAB) andbenign prostatic hyperplasia (BPH). Voiding symptoms comprise hesitancy,poor flow, intermittency, straining and dysuria. Post-micturitionsymptoms comprise terminal dribbling, post-void dribbling and a sense ofincomplete emptying.

Pain and/or other symptoms associated with endometriosis and/or uterinefibroids may comprise dysmenorrhoea; chronic non-menstrual, pelvic pain;dyspareunia; dyschexia; menorrhagia; lower abdominal or back pain;infertility and subfertility; dysuria; bloating and pain on micturition;nausea, vomiting and/or diarrohea. Symptoms may also comprise symptomsrelated to endometriotic lesions or fibroids located outside theperitoneal cavity including for example thoracic endometriosis syndromemanifest as haemoptysis, pneumothorax or haemothorax, and pulmonaryleiomyosis manifest as dyspnoea and a pulmonary mass.

The following examples are provided to illustrate, but not to limit, theinvention. The Examples in WO2004/058184 are referred to illustrate theantibodies for use in the present invention. The entire content ofWO2004/058184 is hereby incorporated by reference.

EXAMPLES Example 1 Antibody Production and Purification

For expression of full antibodies, heavy and light chain variableregions were cloned in two mammalian expression vectors (Eb.911.E3 orEb.pur.911.3E for light chain and Db.911.3E for heavy chain; describedherein) and transfected using lipofectemine into HEK 293 cells fortransient expression. Antibodies were purified using protein A usingstandard methods.

The generation, production, purification and characterization of theanti-NGF antibody E3 is described in the Examples of WO2004/058184, theentire content of which is herein incorporated by reference. Vectorsincorporating E3 light chain and E3 heavy chain have been deposited withthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va., USA (ATCC):

ATCC Accession Date of Material No. Deposit Eb.911.3E E3 light chainPTA-4893 Jan. 8, 2003 Eb.pur.911.3E E3 light chain PTA-4894 Jan. 8, 2003Db.911.3E E3 heavy PTA-4895 Jan. 8, chain 2003

Details concerning these deposits can be found in WO2004058184, thecontent of which is herein incorporated by reference in its entirety.

Three mammalian expression vectors were designed and constructed for usein the expression of antibody E3 in mammalian cells.

Vector Db.911.3E is an expression vector comprising the heavy chainvariable region of the E3 antibody and the human IgG2a constant region,and is suitable for transient or stable expression of the heavy chain.Db.911.3E consists of nucleotide sequences corresponding to thefollowing regions: the murine cytomegalovirus promoter region(nucleotides 1-612); a synthetic intron (nucleotides 619-1507); the DHFRcoding region (nucleotides 707-1267); human growth hormone signalpeptide (nucleotides 1525-1602); antibody 3E heavy chain variable region(nucleotides 1603-1965); human heavy chain IgG2a constant regioncontaining the following mutations: A330P331 to S330S331 (amino acidnumbering with reference to the wildtype IgG2a sequence; see Eur. J.Immunol. (1999) 29:2613-2624); SV40 late polyadenylation signal(nucleotides 2974-3217); SV40 enhancer region (nucleotides 3218-3463);phage f1 region (nucleotides 3551-4006) and beta lactamase (AmpR) codingregion (nucleotides 4443-5300). Db.911.3E was deposited at the ATCC onJan. 8, 2003, and was assigned ATCC Accession No. PTA-4895.

Vector Eb.911.3E is an expression vector comprising the light chainvariable region of the E3 antibody and the human kappa chain constantregion, and is suitable for transient expression of the light chain.Eb.911.3E consists of nucleotide sequences corresponding to thefollowing regions: the murine cytomegalovirus promoter region(nucleotides 1-612); human EF-1 intron (nucleotides 619-1142); humangrowth hormone signal peptide (nucleotides 1173-1150); antibody E3 lightchain variable region (nucleotides 1251-1571); human kappa chainconstant region (nucleotides 1572-1892); SV40 late polyadenylationsignal (nucleotides 1910-2153); SV40 enhancer region (nucleotides2154-2399); phage f1 region (nucleotides 2487-2942) and beta lactamase(AmpR) coding region (nucleotides 3379-4236). Eb.911.3E was deposited atthe ATCC on Jan. 8, 2003, and was assigned ATCC Accession No. PTA-4893.

Vector Eb.pur.911.3E is an expression vector comprising the light chainvariable region of the E3 antibody and the human kappa constant region,and is suitable for stable expression of the light chain. Eb.pur.911.3Econsists of nucleotide sequences corresponding to the following regions:the murine cytomegalovirus promoter region (nucleotides 1-612); humanEF-1 intron (nucleotides 619-1758); pac gene (puromycinR) coding region(nucleotides 739-1235); human hsp70 5′UTR region (nucleotides1771-1973); human growth hormone signal peptide (nucleotides 1985-2062);antibody E3 light chain variable region (nucleotides 2063-2383); humankappa chain constant region (nucleotides 2384-2704); SV40 latepolyadenylation signal (nucleotides 2722-2965); SV40 enhancer region(nucleotides 2966-3211); phage f1 region (nucleotides 3299-3654) andbeta lactamase (AmpR) coding region (nucleotides 4191-5048).Eb.pur.911.E3 was deposited at the ATCC on Jan. 8, 2003, and wasassigned ATCC Accession No. PTA-4894.

Transient cell expression was performed as follows: CHO and HEK293Tcells in 150 mm dishes were transiently co-transfected with 25 ug ofeach plasmid (i.e., one plasmid containing the heavy chain and oneplasmid containing the light chain). DNA was mixed with 100 ullipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. The DNA-lipid complexes were allowed to contact the cellsin DMEM/F12 medium without serum or antibiotics for 5 hours. Followingthis incubation, the media was changed for expression to Opti-MEM(Invitrogen) without any additives for two days. Cell supernatantscontaining antibody were harvested sequentially up to four times withsubsequent media replacement. Supernatants were purified by affinitychromatography using MapSelect Protein A resin (Amersham biosciences17-5199-02). Antibody was bound to the protein A resin in 0.3M glycine,0.6M NaCl buffer at pH 8, then eluted with 0.1 M citrate buffer at pH 3.Fractions containing antibody were immediately neutralized with 1 M Trisbuffer at pH 8.0, Antibody fractions were then dialyzed and concentratedin PBS. Antibodies were selected, and assayed as follows:

Biacore Assay

Affinities of anti-NGF Fabs and monoclonal antibodies were determinedusing the BIAcore3000™ surface plasmon resonance (SPR) system (BIAcore,INC, Piscaway N.J.). CM5 chips were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Human NGF was diluted into 10 mM sodium acetate pH 4.0 and injected overthe activated chip at a concentration of 0.005 mg/mL. Using variableflow time across the individual chip channels, two ranges of antigendensity were achieved: 100-200 response units (RU) for detailed kineticstudies and 500-600 RU for screening assays. The chip was blocked withethanolamine. Regeneration studies showed that a mixture of Pierceelution buffer (Product No. 21004, Pierce Biotechnology, Rockford, Ill.)and 4 M NaCl (2:1) effectively removed the bound Fab while keeping theactivity of hNGF on the chip for over 200 injections. HBS-EP buffer(0.01M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% Surfactant P29) wasused as running buffer for all the BIAcore assays.

Screening Assay

A screening BIAcore assay was optimized to determine the affinity of Fabclones from libraries. Supernatants of small culture lysates wereinjected at 50 μl/min for 2 min. Dissociation times of 10 to 15 minuteswere used for determination of a single exponential dissociation rate(k_(off)) using BIAevaluation software. Samples that showed k_(off)rates in the same range as the template used to create the library(clone 8L2-6D5, k_(off) 1×10⁻³ s⁻¹) were injected for confirmation anddissociation times of up to 45 min were allowed to obtain better k_(off)values. Clones showing improved (slower) k_(off) values were expressedat large scale and full kinetic parameters, k_(on) and k_(off), weredetermined on purified protein. The assay was capable of detectingdifferences in affinity that were approximately 2-fold or larger.

Affinity Determination Assay

Serial dilutions (0.1-10× estimated K_(D)) of purified Fab samples wereinjected for 1 min at 100 μL/min and dissociation times of up to 2 hwere allowed. The concentrations of the Fab proteins were determined byELISA and/or SDS-PAGE electrophoresis using as a standard a Fab of knownconcentration (as determined by amino acid analysis). Kineticassociation rates (k_(on)) and dissociation rates (k_(off)) wereobtained simultaneously by fitting the data to a 1:1 Langmuir bindingmodel (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values were calculated as k_(off)/k_(on).

Example 2 Analysis of Buffers and pH

A study was conducted to evaluate the effect of four different bufferson antibody aggregation and fragmentation.

Specifically, four liquid formulations comprising anti-NGF antibody E3and buffered with acetate, succinate, histidine or Citrate wereprepared. The formulations then were stored at 5, 25 and 40° C. andantibody aggregation, fragmentation and oxidation measurements weretaken at 0, 4, 9 and 13 weeks.

The manufacturing process can be summarized as follows: The buffer isprepared, pH adjusted, and sterile filtered (see Table 2.1 and 2.2 fordetails). The antibody is concentrated then buffer exchanged. Theantibody is analyzed with UV and then diluted with respective buffer to20 mg/mL. The 20 mg/mL solution is then sterile filtered. Finally, thesterile solution is filled, stoppered and capped with an overseal.

Aggregation Analysis:

The antibody formulations of Table 2.2 were stored at a temperature of5, 25 and 40° C. for 0, 4, 9 and 13 weeks.

Each formulation was analyzed for aggregation using size exclusionchromatography (SEC). The size exclusion chromatography was carried outusing a TSK gel G3000SWXL-G2000SWXL column, mobile phase 0.2 M sodiumphosphate buffer at pH 7.0, a flow rate of 1 ml/min, and UV detection at214 nm.

Aggregation levels were calculated by integrating the areas under thechromatogram peaks for each formulation and reporting the integratedareas under the high molecular weight species peaks as a percentage oftotal peak area. As can be seen in Table 2.3, the histidine-bufferedformulations particularly at pH 6, showed the lowest levels ofaggregation, followed by the acetate, succinate, then citrate bufferedformulations, in that order.

Fragmentation Analysis:

The antibody formulations of Table 2.2 were stored at a temperature of5, 25 and 40° C. for 0, 4, 9 and 13 weeks.

Each formulation also was analyzed for fragmentation using reducedcapillary gel electrophoresis (rCGE). Proteins unfold (denature) andbecome ‘rod-like’ in structure following the cleavage of it's disulphidebonds in red-CGE. The ‘reduced’ protein separates into its heavy andlight chains allowing for their quantization along with fragmentedspecies. Reduced-CGE is considered a reliable method for quantifyingpercent fragments (% impurities). The percentage of fragmentation wasmeasured at the relevant times for each of the formulations. Thefragmentation levels were calculated as a percentage of total bandvolume. As can be seen in Table 2.4, the histidine-buffered formulationsparticularly at pH 6, showed the lowest levels of fragmentation.

Oxidation Analysis:

Oxidation levels of methionine residues at amino acid positions X and Yin anti-NGF antibody E3 were measured by a Lysine-C mapping method afterstorage for at 5, 25 and 40° C. for 0, 4, 9 and 13 weeks. Samples ofeach formulation tested were then digested with Lyc-C enzyme in trisbuffer at pH 8.0 under standard conditions and analyzed byreversed-phase high performance liquid chromatography. Separation wasaccomplished using a Grace Vydae Protein C4 analytical column with 0.1%TFA in water and 0.085% TFA in Acetonitrile gradient elution. Percentoxidation of methionine amino acids in Anti-NGF antibody E3 wasrecorded.

The results in Table 2.5 indicate that the percent oxidation of thecitrate formulation is greatest.

TABLE 2.1 Buffer Preparation Table: Summary of recorded amounts ofbuffer components from manufacturing records for compounding 250 mL ofbuffer. Formulation Acetate Acetic Acid Histidine Histidine CitrateCitric Acid Succinate Succinic Acid Lot Number pH (mg/mL) (mg/mL)(mg/mL) HCl (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) Acetate pH 5.0 5.0435.58 104.14 — — — — — — Acetate pH 5.5 5.5 577.75 34.59 — — — — — —Acetate pH 6.0 6.0 644.28 15.07 — — — — — — Acetate pH 6.5 6.5 668.5110.32 — — — — — — Histidine pH 5.0 5.0 — — 70.45 839.27 — — — —Histidine pH 5.5 5.5 — — 186.9 751.9 — — — — Histidine pH 6.0 6.0 — —387.5 454.1 — — — — Histidine pH 6.5 6.5 — — 588.75 220.87 — — — —Citrate pH 5.0 5.0 — — — — 233.63 107.18 — — Citrate pH 5.5 5.5 — — — —279.09 79.01 — — Citrate pH 6.0 6.0 — — — — 321.25 42.51 — — Citrate pH6.5 6.5 — — — — 348.77 20.52 — — Succinate pH 5.0 5.0 — — — — — — 585.18176.86 Succinate pH 5.5 5.5 — — — — — — 679.4 80.83 Succinate pH 6.0 6.0— — — — — — 756.23 34.73 Succinate pH 6.5 6.5 — — — — — — 791.83 11.60

TABLE 2.2 Formulation Matrix Table: Formulation mAb Con Acetate AceticAcid Histidine Histidine HCl Citrate Citric Acid Succinate Succinic AcidLot Number pH (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL)(mg/mL) (mg/mL) Acetate pH 5.0 5.0 20 1.7423 0.4323 — — — — — — AcetatepH 5.5 5.5 20 2.3110 0.1813 — — — — — — Acetate pH 6.0 6.0 20 2.57710.0639 — — — — — — Acetate pH 6.5 6.5 20 2.6744 0.0210 — — — — — —Histidine pH 5.0 5.0 20 — — 0.2818 3.8182 — — — — Histidine pH 5.5 5.520 — — 0.7448 3.1909 — — — — Histidine pH 6.0 6.0 20 — — 1.5500 2.1000 —— — — Histidine pH 6.5 6.5 20 — — 2.3552 1.0091 — — — — Citrate pH 5.05.0 5 — — — — 0.9345 0.3821 — — Citrate pH 5.5 5.5 5 — — — — 1.11640.2530 — — Citrate pH 6.0 6.0 5 — — — — 1.2850 0.1325 — — Citrate pH 6.56.5 5 — — — — 1.3951 0.0538 — — Succinate pH 5.0 5.0 20 — — — — — —2.3407 0.6561 Succinate pH 5.5 5.5 20 — — — — — — 2.7183 0.3809Succinate pH 6.0 6.0 20 — — — — — — 3.0249 0.1575 Succinate pH 6.5 6.520 — — — — — — 3.1673 0.0537

TABLE 2.3 Summary of % Aggregation (via SEC) Data from 20 mg/mL anti-NGFantibody: pH and Buffer Screening Study Condition 5° C. 25° C. 40° C.Weeks Lot # 0 13 0 9 13 0 4 9 13 Acetate pH 5.0 1.6 1.8 1.6 2.2 2.5 1.63.2 5.1 8.1 Acetate pH 5.5 1.8 2.1 1.8 2.7 3.0 1.8 3.4 4.9 7.1 AcetatepH 6.0 2.0 2.4 2.0 3.2 3.3 2.0 4.3 5.5 7.0 Acetate pH 6.5 2.0 2.5 2.03.5 3.9 2.0 4.4 5.8 7.0 Histidine pH 5.0 1.6 1.8 1.6 2.0 2.3 1.6 2.7 3.97.0 Histidine pH 5.5 1.6 1.8 1.6 2.2 2.4 1.6 2.7 3.4 5.1 Histidine pH6.0 1.8 2.1 1.8 2.5 2.7 1.8 3.0 3.5 4.8 Histidine pH 6.5 1.9 2.2 1.9 2.83.1 1.9 3.4 4.2 5.6 Citrate pH 5.0 1.6 1.9 1.6 2.4 2.8 1.6 4.6 8.5 15.0Citrate pH 5.5 1.8 2.2 1.8 3.0 3.3 1.8 4.9 8.7 12.2 Citrate pH 6.0 2.02.5 2.0 3.3 3.7 2.0 4.4 6.5 8.4 Citrate pH 6.5 2.1 2.8 2.1 3.7 4.2 2.14.8 6.5 8.4 Succinate pH 5.0 1.6 1.9 1.6 2.3 2.7 1.6 3.5 6.1 11.2Succinate pH 5.5 2.0 2.2 2.0 2.8 3.2 2.0 3.9 5.7 8.4 Succinate pH 6.01.8 2.5 1.8 3.1 3.6 1.8 4.3 5.8 7.8 Succinate pH 6.5 2.2 2.8 2.2 3.7 4.12.2 4.8 5.9 7.5

TABLE 2.4 Summary of % Fragmentation (via reduced CGE) Data from 20mg/mL anti-NGF antibody: pH and Buffer Screening Study Condition 5 C. 25C. 40 C. Weeks Lot # 0 13 0 9 13 0 4 9 13 Acetate pH 5.0 1.1 1.1 1.1 1.41.0 1.1 4.2 7.0 10.3 Acetate pH 5.5 1.1 1.0 1.1 1.0 1.7 1.1 4.4 5.2 9.4Acetate pH 6.0 1.1 1.1 1.1 1.4 1.3 1.1 3.6 5.9 7.2 Acetate pH 6.5 1.11.2 1.1 1.0 2.2 1.1 4.1 6.4 8.1 Histidine pH 5.0 1.1 1.0 1.1 1.0 1.8 1.15.8 7.5 11.2 Histidine pH 5.5 1.1 1.1 1.1 0.9 1.5 1.1 4.3 6.4 7.9Histidine pH 6.0 1.1 1.0 1.1 1.6 1.5 1.1 3.1 3.8 6.1 Histidine pH 6.51.0 1.2 1.0 1.6 2.2 1.0 3.7 6.1 9.7 Citrate pH 5.0 1.0 1.0 1.0 1.0 2.11.0 5.4 9.1 14.5 Citrate pH 5.5 1.0 1.1 1.0 1.1 1.4 1.0 4.6 7.6 11.5Citrate pH 6.0 1.1 1.6 1.1 2.0 1.6 1.1 3.7 6.9 10.6 Citrate pH 6.5 1.51.0 1.5 1.3 9.1 1.5 3.4 8.2 10.9 Succinate pH 5.0 1.2 1.0 1.2 2.1 7.91.2 5.4 8.8 13.1 Succinate pH 5.5 1.2 1.5 1.2 0.9 0.9 1.2 3.1 5.8 9.0Succinate pH 6.0 1.2 1.0 1.2 1.3 6.8 1.2 3 5.0 8.0 Succinate pH 6.5 1.21.1 1.2 1.5 11.8 1.2 3.5 7.2 8.0

TABLE 2.5 Summary of % Oxidation Data from 20 mg/mL anti-NGF antibody:pH and Buffer Screening Study Condition 5 C. 25 C. 40 C. Weeks Lot # 0 913 0 9 13 0 4 9 13 Acetate pH 5.0 2.8 2.1 2.8 2.3 2.8 3.3 4.1 Acetate pH5.5 2.8 2.1 2.8 2.3 2.8 3.3 4.1 Acetate pH 6.0 2.3 2.0 2.3 2.2 2.3 3.13.4 Acetate pH 6.5 2.3 2.1 2.3 2.5 2.3 3.5 4.1 Histidine pH 5.0 2.2 2.02.2 2.5 2.2 4.1 6.4 Histidine pH 5.5 2.6 1.8 2.6 2.3 2.6 3.9 5.7Histidine pH 6.0 2.0 1.9 2.0 2.3 2.0 3.9 6.1 Histidine pH 6.5 2.4 2.12.4 2.5 2.4 4.7 7.1 Citrate pH 5.0 2.1 2.1 2.1 2.8 2.1 7.2 9.7 CitratepH 5.5 2.8 2.1 2.8 2.9 2.8 8.0 10.1 Citrate pH 6.0 2.4 1.8 2.4 2.7 2.46.6 7.9 Citrate pH 6.5 2.4 1.8 2.4 2.6 2.4 6.3 8.1 Succinate pH 5.0 3.91.8 3.9 2.2 3.9 4.0 5.5 Succinate pH 5.5 2.7 1.9 2.7 2.3 2.7 4.1 4.6Succinate pH 6.0 2.1 2.1 2.1 2.1 2.1 3.8 3.4 Succinate pH 6.5 2.7 2.12.7 2.1 2.7 3.5 3.1

Example 3 Analysis of Tonicity Agents. Trehalose Vs Sucrose Vs Sorbitol

A study was conducted to compare the effect of tonicity agents inparticular trehalose, sucrose and sorbitol on stability of anti-NGFantibody E3 formulations.

Specifically, three liquid formulations comprising anti-NGF antibody E3and trehalose, sucrose or sorbitol were prepared as listed in Table 3.1.The formulations then were stored at 5, 25 and 40° C. and antibodyaggregation, fragmentation and oxidation measurements were taken at 2,4, 8, 13 and 26 weeks.

The manufacturing process for formulations in Table 3.1 can besummarized as follows: The buffer is prepared, pH adjusted, and sterilefiltered (see Table 3.1 for details). Stock excipients solutions areprepared and sterile filtered. The antibody is concentrated then bufferexchanged. The concentrated antibody is analyzed with UV and thendiluted with respective buffer to 50 mg/mL and respective excipientscombined to the required concentration. The 50 mg/mL solution is thensterile filtered. Finally, the sterile solution is filled, stoppered andoversealed. All formulations have a pH of 6.0 and an anti-NGF antibodyE3 concentration of 50 mg/ml.

Aggregation Analysis:

The antibody formulations of Table 3.1 were stored at a temperature of5, 25 and 40° C. for 2, 4, 8, 13 and 26 weeks.

Each formulation was analyzed for aggregation using the method describedin Example 2. Aggregation levels were calculated and are shown in FIG.1.

Fragmentation Analysis:

The antibody formulations of Table 3.1 were stored at a temperature of5, 25 and 40° C. for 2, 4, 8, 13 and 26 weeks.

Each formulation also was analyzed for fragmentation using themethodology of Example 2. As can be seen in FIG. 2, the trehaloseformulation, showed the lowest levels of fragmentation at 13 weeksstorage.

TABLE 3.1 Formulation Matrix Formulation Lot mAbConc Histidine TrehaloseSucrose Sorbitol Number pH (mg/mL) (mM) (mg/mL) (mg/mL) (mg/mL)114069-001-A# 6.0 50 10 84 — — 114069-001-B# 6.0 50 10 — 80 —114069-001-C# 6.0 50 10 — — 40

Example 4 Tonicity Agent Screening Study: Trehalose vs. Sucrose

A study was conducted to compare the effect of tonicity agents inparticular trehalose and sucrose on stability and activity of anti-NGFantibody E3 formulations. The hydrolysis of sucrose to fructose andglucose in a dilute acid solution is well known. Glucose molecules arealso known to bind at random with the lysine residues of a protein'samino acid sequence. This is known as glycation. Therefore a proteinformulation, buffered to an acidic pH, containing sucrose, couldexperience sucrose hydrolysis and then glycation. The glycated proteincould undergo degradation processes more readily than an unglycatedprotein. Hence, the presence of sucrose in a liquid protein formulationcould have an adverse impact on the protein's quality over itsshelf-life. In contrast to this, trehalose is not known to undergo sucha hydrolysis-based degradation and can be a preferential tonicitymodifying agent in protein formulations.

Specifically, six liquid formulations comprising anti-NGF antibody E3with sucrose and two liquid formulations comprising anti-NGF antibody E3and trehalose were prepared, see Table 4.1. The formulations then werestored at 5, 25 and 40° C. and antibody aggregation, and glycationmeasurements were taken at 2, 4, 8, 13 and 26 weeks.

The manufacturing process for formulations in Table 4.1 can besummarized as follows: The buffer is prepared, pH adjusted, and sterilefiltered (see Table 4.1 for details). Stock excipients solutions areprepared and sterile filtered. The antibody is concentrated then bufferexchanged. The concentrated antibody is analyzed with UV and thendiluted with respective buffer to 10 mg/ml or 50 mg/ml and respectiveexcipients combined to the required concentration. The 10 mg/ml or 50mg/ml solution is then sterile filtered. Finally, the sterile solutionis filled, stoppered and oversealed.

Aggregation Analysis:

The antibody formulations of Table 4.1 were stored at a temperature of5, 25, 40 and 50° C. for 2, 4, 8, 13 and 26 weeks.

Each formulation was analyzed for aggregation using size exclusionchromatography (SEC) as described in Example 2. Calculated aggregationlevels are compared in Table 4.2 and show that the trehalosedemonstrates as low if not lower levels of aggregation compared tosucrose for the same antibody concentrations investigated.

Glycation Analysis:

Glycation levels of lysine residues in anti-NGF antibody E3 weremeasured by a mass spectrometry mapping method after storage of theformulations at 25, 40 and 50° C. for 0, 2, 4, 8 and 13 weeks forformulation samples comprising either sucrose or trehalose tonicityagent according to Table 4.1 (10 mg/ml antibody, 10 mM Histidine pH6.0).Glycation was found to be proportional to the rate of hydrolysis of thetonicity agent. There was no demonstrated hydrolysis of trehalose, hencetrehalose is a preferred tonicity agent over sucrose for the antibodyformulation as glycation of the formulation antibody is avoided, sucrosedemonstrated between 1% and 2% hydrolysis in formulations stored at 25°C. for 104 weeks.

TABLE 4.1 Formulation Matrix Table for Example 4 Formulation mAbConcHistidine Histidine HCl Sucrose Trehalose Glucose Fructrose MethionineEDTA PS20 Lot Number pH (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL)(mg/mL) (mg/mL) (mg/mL) (mg/mL) 112633-174 6.0 10 0.8206 0.9879 94 — — —— — 0.1 112633-175 6.0 10 0.8206 0.9879 94 — — — — 0.05 0.1 112633-1766.0 10 0.8206 0.9879 94 — — — 0.1 0.05 0.1 112633-177 6.0 10 0.82060.9879 94 — — — 0.1 — 0.1 112633-178 5.7 10 0.5586 1.3418 94 — — — — —0.1 112633-179 5.7 10 0.5586 1.3418 94 — — — — 0.05 0.1 112633-180 6.010 0.8206 0.9879 — 84 — — — 0.05 0.1 112633-181 5.7 10 0.5586 1.3418 —84 — — — 0.05 0.1 112633-182 6.0 10 0.8206 1.3418 — — 49.5 49.5 — 0.050.1

TABLE 4.2 Summary of % Aggregation (via SEC) Data from 10 mg/mL anti-NGFantibody: Comparison of sucrose vs. trehalose Condition 25° C. 40° C.50° C. weeks Lot# 0 4 12 0 4 12 0 4 12 112633-174 0.5 0.5 0.8 0.5 1.01.9 0.5 4.3 20.4 112633-175 0.5 0.6 0.6 0.5 0.7 1.4 0.5 3.2 19.2112633-176 0.5 0.6 0.7 0.5 0.7 1.2 0.5 2.9 13.5 112633-177 0.5 0.6 0.60.5 0.8 1.2 0.5 2.8 13.2 112633-178 0.5 0.5 0.6 0.5 1.0 1.8 0.5 4.6 24.8112633-179 0.5 0.5 0.6 0.5 0.7 1.4 0.5 3.6 20.0 112633-180 0.5 0.6 0.70.5 0.8 1.6 0.5 3.1 11.4 112633-181 0.5 0.5 0.6 0.5 0.7 1.7 0.5 3.7 12.5112633-182 0.5 0.6 0.9 0.5 1.7 12.1 0.5

TABLE 4.3 Summary of Glycation of the anti-NGF antibody (reported as %heavy chain + 162 Daltons) Data from 10 mg/mL anti-NGF antibody:Comaprison of sucrose vs. trehalose Condition 25° C. 40° C. 50° C. weeksLot# 0 4 12 0 4 12 0 4 9 12 112633- 0 — — 0 — 4 0 3 19 23 174 112633- 0— — 0 — 4 0 4 22 29 175 112633- 0 — — 0 — 3 0 4 13 17 176 112633- 0 — —0 — 3 0 4 14 18 177 112633- 0 — — 0 — 4 0 7 29 49 178 112633- 0 — — 0 —4 0 7 30 52 179 112633- 0 — — 0 — — 0 — — — 180 112633- 0 — — 0 — — 0 1— — 181 112633- 19 67 — 19 92 60 19 100 182

Example 5 Analysis of Surfactant Agents and Polymer Stabilizers

A study was conducted to compare the effect of surfactants and polymerstabilizers, in particular PS20, PS80, PEG3350, PEG3350+PS20 onstability of anti-NGF antibody E3 formulations.

Specifically, four liquid formulations comprising anti-NGF antibody E3and PS20, PS80, PEG3350, PEG3350+PS20 were prepared as listed in Table5.1. The formulations then were stored at 5, 25 and 40° C. and antibodyaggregation, fragmentation and oxidation measurements were taken at 2,4, 8, 13 and 26 weeks. All formulations had a pH of 6.0 and an anti-NGFantibody E3 concentration of 50 mg/ml. The manufacturing process forformulations in Table 5.1 can be summarized as follows: The buffer isprepared, pH adjusted, and sterile filtered (see Table 5.1 for details).Stock excipients solutions are prepared and sterile filtered. Theantibody is concentrated then buffer exchanged. The concentratedantibody is analyzed with UV and then diluted with respective buffer to50 mg/ml and respective excipients combined to the requiredconcentration. The 50 mg/ml solution is then sterile filtered. Finally,the sterile solution is filled, stoppered and oversealed. Tables 5.2 and5.3 report the results.

Aggregation Analysis:

The antibody formulations of Table 5.1 were stored at a temperature of5, 25 and 40° C. for 2, 4, 8, 13 and 26 weeks.

Each formulation was analyzed for aggregation using size exclusionchromatography (SEC) as described in Example 2. Calculated aggregationlevels are compared in Table 5.2 and show that the PS20 at aconcentration of 0.2 mg/ml demonstrates equivalent levels of aggregationcompared to PS80 of 0.2 mg/ml and PEG3350 1 mg/ml investigated. PS20 ata concentration of 0.1 mg/ml also demonstrates equivalent results (datafro 40° C.).

Fragmentation Analysis:

The antibody formulations of Table 5.1 were stored at a temperature of5, 25 and 40° C. for 2, 4, 8, 13 and 26 weeks.

Each formulation was analyzed for fragmentation using the methodology ofExample 2. Data collected from the 40° C. samples is shown in Table 5.3,and show that the PS20 at a concentration of 0.2 mg/ml demonstratesequivalent levels of fragmentation compared to PS80 of 0.2 mg/ml andPEG3350 1 mg/ml investigated. PS20 at a concentration of 0.1 mg/ml wasalso shown to demonstrate equivalent results.

TABLE 5.1 Formulation Matrix for Surfactant Agent Screening StudyFormulation mAbConc Histidine PEG 3350 PS20 + PEG Lot Number pH (mg/mL)(mM) PS20 (mg/mL) PS80 (mg/mL) (mg/mL) 3350 114069-001-D# 6.0 50 10 0.2— — — 114069-001-E# 6.0 50 10 — 0.2 — — 114069-001-F# 6.0 50 10 — — 10 —114069-001-G# 6.0 50 10 — — — 0.2 + 1

TABLE 5.2 Summary of % Aggregation (via SEC) Data from 50 mg/mL anti-NGFantibody: Surfactant Agent Screening Study 25 C. 40 C. Weeks T0 4 8 1326 2 4 8 13 26 114069-001- PS20 1 1.4 1.5 1.8 2.4 1.8 2.2 2.8 4.2 8.2 D2114069-001- PS80 0.9 1.3 1.5 1.8 n/a 1.8 2.2 3.1 4.2 n/a E2 114069-001-PEG3350 1 1.4 1.5 1.9 n/a 1.8 2.2 2.8 3.9 n/a F2 114069-001- PS20 + 0.91.3 1.5 1.9 n/a 1.8 2.1 3 4.3 n/a G2 PEG3350

TABLE 5.3 Summary of % Fragments (via reduced CGE) Data from 50 mg/mLanti-NGF antibody: Surfactant Screening Study 25 C. 40 C. Weeks T0 4 813 26 2 4 8 13 26 114069- PS20 0.9 0.8 1.3 2.3 3.1 1.3 2.6 5.5 7.1 13.6001-D2 114069- PS80 0.9 0.7 0.9 1.7 n/a 1.4 1.7 6.4 6.5 n/a 001-E2114069- PEG3350 0.9 0.8 1.6 2.3 n/a 1.6 1.8 4.5 7 n/a 001-F2 114069-PS20 + 0.8 0.7 1 2.3 n/a 1.3 1.2 4.9 7.9 n/a 001-G2 PEG3350

Example 6: Analysis of Anti-Oxidant Agents

A study was conducted to compare the effect of anti-oxidant agents inparticular methionine on stability of anti-NGF antibody E3 formulations.

Specifically, nine liquid formulations comprising anti-NGF antibody E3with and without methionine were prepared. All formulations had a pH of6.0 and an anti-NGF antibody E3 concentration of 10 or 50 mg/ml, 84mg/ml trehalose or sucrose and 0.1 mg/ml PS20, 10 mM histidine, plus orminus 0.05 mg/ml EDTA.

The manufacturing process for formulations can be summarized as follows:The buffer is prepared, pH adjusted, and sterile filtered. Stockexcipients solutions are prepared and sterile filtered. The antibody isconcentrated then buffer exchanged. The concentrated antibody isanalyzed with UV and then diluted with respective buffer to 10 or 50mg/ml and respective excipients combined to the required concentration.The 10 mg/ml or 50 mg/ml solution is then sterile filtered. Finally, thesterile solution is filled, stoppered and oversealed.

The formulations then were stored at 5, 25 and 40° C. and antibodyaggregation, fragmentation and oxidation measurements were taken at 2,4, 8, 13, 26 and 52 weeks.

Aggregation Analysis:

The antibody formulations of were stored at a temperature of 5, 25 and40° C. for 0, 8, 13, 26 and 52 weeks.

Each formulation was analyzed for aggregation using size exclusionchromatography (SEC) as described in Example 2. Calculated aggregationlevels were compared and found not to be significantly different forsamples stored at 5 or 25° C. for 26 weeks with or without methionine,the presence of methionine has no discernable effect on aggregation.

Fragmentation Analysis:

The antibody formulations were stored at a temperature of 5, 25 and 40°C. for 0, 8, 13 and 26 weeks, fragmentation measures were taken at 26weeks.

Each formulation was analyzed for fragmentation using the methodology ofExample 2. Calculated fragmentation levels were compared and found notto be significantly different for samples stored at 5 or 25° C. for 26weeks with or without methionine, the presence of methionine has nodiscernable effect on fragmentation.

Oxidation Analysis:

Oxidation levels of methionine residues at amino acid positions X and Yin anti-NGF antibody E3 were measured by a Lysine-C mapping method afterstorage for at 5, 25 and 40° C. for 0, 13, 26 and 52 weeks. Percentoxidation of methionine amino acids in Anti-NGF antibody E3 was recordedas described in Example 2.

The results in Table 6.1 indicate that the percent oxidation of theantibody is reduced by the presence of methionine under longer periodsof storage.

TABLE 6.1 Summary of % Oxidation Data from 10 and 50 mg/mL anti-NGFantibody: Antioxidant Agent Screening Study 25° C. 40° C. Weeks 0 13 2652 13 26 52 10 mM Histidine + Suc + PS20 + 10 mg/mL 3.50 3.40 4.70 7.8021.00 51.60 mAb 10 mM Histidine + Suc + PS20 + 4.70 3.20 0.00 7.00 10.900.00 EDTA + Met + 10 mg/mL mAb 10 mM Histidine + Suc + PS20 + 50 mg/mL1.77 3.90 3.80 5.50 8.40 15.50 39.20 mAb 10 mM Histidine + Suc + PS20 +1.72 2.70 3.40 3.10 5.10 10.10 30.60 EDTA + Met + 50 mg/mL mAb 10 mMHistidine + Tre + PS20 + 50 mg/mL 1.79 3.40 3.80 5.80 6.80 15.00 30.00mAb 10 mM Histidine + Tre + PS20 + 1.65 3.30 4.00 5.90 6.60 11.20 20.20EDTA + 50 mg/mL mAb 10 mM Histidine + Tre + PS20 + 1.72 2.70 2.80 2.603.90 6.50 14.40 Met + 50 mg/mL mAb 10 mM Histidine + Tre + PS20 + 1.702.70 3.30 3.10 4.60 7.00 14.00 EDTA + Met + 50 mg/mL mAb 10 mMHistidine + Tre + PS20 + 0.00 4.10 3.30 3.30 6.20 6.80 12.60 EDTA +Met + 10 mg/mL mAb

Example 7 Freeze Thaw Stability Study with and without Trehalose

Antibody formulations were prepared comprising 50 mg/ml anti-NGFantibody E3, 10 mM histidine buffer pH 6.0, and 84 mg/ml trehalose,identical samples were prepared lacking trehalose. The samples weresubjected to up to 4 cycles of freezing and thawing and aggregation wasdetermined for the samples. Freeze thaw cycles are 72 hrs freezing at−70° C. and 48 hrs thawing at 5° C.

Each formulation was analyzed for aggregation using size exclusionchromatography (SEC) as described in Example 2. Calculated aggregationlevels are compared in Table 7.1 and show that the samples comprisingtrehalose offer complete protection from freeze thaw effects onaggregation of the antibody.

Samples with trehalose show no appreciable increase in level ofaggregation over time.

TABLE 7.1 Summary of % Aggregation (via SEC) Data from 50 mg/mL anti-NGF antibody: % HMMS of Control % HMMS of Trehalose Freeze/Thaw CycleSample (no Trehalose) Sample 0 0.60 0.60 1 0.75 0.60 2 0.90 0.60 3 1.000.60 4 1.30 0.60

Example 8 Multiple Freeze Thaw Stability of Anti-NGF Antibody E3Formulation with Trehalose

Antibody formulations (112746-124 and 112746-125) were preparedcomprising 22 mg/ml anti-NGF antibody E3, 10 mM histidine buffer pH 6.0,and 84 mg/ml trehalose, 0.05 mg/mL disodium EDTA and 0.1 mg/mLpolysorbate 20. The samples were subjected to up to 15 cycles offreezing and thawing and aggregation was determined for the samples.

Each formulation was analyzed for aggregation using size exclusionchromatography (SEC) as described in Example 2. Calculated aggregationlevels are compared in FIG. 3 and show that all the samples comprisingtrehalose offer complete protection from freeze thaw effects onaggregation of the antibody.

Samples with trehalose show no appreciable increase in level ofaggregation over time.

Example 9: Long-Term Stability Study at 10 and 50 Mg/Ml Anti-NGFAntibody E3: Effect of Trehalose as a Stabilizer

Antibody formulations were prepared comprising 10 mg/ml anti-NGFantibody E3, 10 mM histidine buffer pH 6.0, and 84 mg/ml trehalose,identical samples were prepared lacking trehalose. Details of thesamples studied are given in Table 9.1. The samples were stored at atemperature of 5, 25 and 40° C. for 2, 4, 8, 13, 26 and 52 weeks andaggregation was determined for the samples. Each formulation wasanalyzed for aggregation using size exclusion chromatography (SEC) asdescribed in Example 2. Data collected showed that the samplescomprising trehalose offers high levels of protection from the effectson aggregation of the antibody on storage at accelerated condition of40° C.

TABLE 9.1 Formulation Matrix for Long-Term Stability Study at 10 and 50mg/mL anti-NGF antibody E3: Effect of Trehalose as a stabilizer PF-Formulation 04383119 Histidine Sucrose Trehalose PS20 Methionine EDTALot Number pH (mg/mL) mM (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL)112746-27-1 6.0 10 10 94 — 0.1 — — 112746-27-2 6.0 10 10 94 — 0.1 0.10.05 112746-27-3 6.0 50 10 94 — 0.1 — — 112746-27-4 6.0 50 10 94 — 0.10.1 0.05 112746-27-5 6.0 50 10 — 84 0.1 — — 112746-27-6 6.0 50 10 — 840.1 — 0.05 112746-27-7 6.0 50 10 — 84 0.1 0.1 — 112746-27-8 6.0 50 10 —84 0.1 0.1 0.05 112746-27-9 6.0 10 10 — 84 0.1 0.1 0.05

TABLE 9.2 % Aggregation Data (per SEC) at 40° C. for Long-Term StabilityStudy at 10 and 50 mg/mL anti-NGF antibody E3: Effect of Trehalose as astabilizer Time (weeks) Composition Sample ID 0 2 4 8 13 26 52 10 mMHistidine + Suc + 112746- 0.60 0.70 0.90 1.2 1.7 4.80 22.10 PS20 + 10mg/mL 27-1 mAB 10 mM Histidine + Suc + 112746- 0.60 0.70 0.90 1.2 1.53.40 NA PS20 + EDTA + Met + 27-2 10 mg/mL mAB 10 mM Histidine + Suc +112746- 0.60 1.60 2.00 2.8 3.7 7.50 20.70 PS20 + 50 mg/mL 27-3 mAB 10 mMHistidine + Suc + 112746- 0.60 1.50 1.80 2.4 3.2 6.00 22.40 PS20 +EDTA + Met + 27-4 50 mg/mL mAB 10 mM Histidine + Tre + 112746- 0.60 1.701.90 2.9 3.8 7.60 16.20 PS20 + 50 mg/mL 27-5 mAB 10 mM Histidine + Tre +112746- 0.60 1.70 2.00 2.6 3.3 5.40 13.20 PS20 + EDTA + 50 mg/mL 27-6mAB 10 mM Histidine + Tre + 112746- 0.60 1.60 1.80 2.5 3.2 5.40 12.60PS20 + Met + 50 mg/mL 27-7 mAB 10 mM Histidine + Tre + 112746- 0.60 1.601.80 2.5 3.1 4.80 11.60 PS20 + EDTA + Met + 27-8 50 mg/mL mAB 10 mMHistidine + Tre + 112746- 0.60 0.90 1.00 1.4 1.8 3.10  7.00 PS20 +EDTA + Met + 27-9 10 mg/mL mAB

Example 10: Long-Term Stability Study of Anti-NGF Antibody E3Formulation Containing Trehalose, EDTA, Polysorbate 20 in HistidineBuffer at pH 6.0 at 2.5, 5, 10, 20 and 50 mg/mL

Antibody formulations were prepared comprising 2.5, or 5 or 10 or 20 or50 mg/mL anti-NGF antibody E3, 10 mM histidine buffer pH 6.0, and 84mg/ml trehalose, 0.05 mg/mL EDTA and 0.1 mg/mL polysorbate 20. Thesamples were stored at a temperature of between 5 and 8° C. for up to 24months and beyond and aggregation, fragmentation, oxidation wasdetermined for the samples.

Data collected and presented in FIGS. 4, 5, and 6 indicate that the saidformulation of anti-NFG antibody E3 at 2.5, 5, 10, 20 or 50 mg/mL mAbconcentration is stable for up to 52 weeks.

An example of a liquid antibody composition according to the presentinvention is as follows:

any of about 2.5 mg/ml, 5 mg/ml, 10 mg/ml, 20 mg/ml, 22 mg/ml or about50 mg/ml of an antibody comprising a variable heavy chain sequence ofSEQ ID NO. 1 and a variable light chain sequence of SEQ ID NO. 2,

about 10 mM histidine buffer,

about 84 mg/ml trehalose dihydrate,

about 0.01 weight/volume Polysorbate 20,

about 0.005% disodium EDTA,

wherein said composition is of a pH 6.0+/−0.2. The liquid antibodycomposition is preferably of a total volume of around 1 ml.

Antibody Sequences

Heavy chain variable region E3 (Kabat CDRs are underlined; Chothia CDRsare BOLD AND ITALICIZED)

(SEQ ID NO: 1) QVQLQESGPGLVKPSETLSLTCTVSGFSLI

WIRQPPGKGLEWIG

RVTISKDTSKNQFSLKLSSVTAADTAVYYCAR

WGQGTLVTVS

Light chain variable region E3 (Kabat CDRs are underlined; Chothia CDRsare BOLD AND ITALICIZED)

(SEQ ID NO: 2) DIQMTQSPSSLSASVGDRVTITC

WYQQKPGKAPKLLIY

VPSRFSGSGSGTDFTFTISSLQPEDIATYYC

F GQGTKLEIKRT E3 heavy chain extended CDRs (SEQ ID NO: 3)CDRH1: GFSLIGYDLN (SEQ ID NO: 4) CDRH2: IIWGDGTTDYNSAVKS (SEQ ID NO: 5)CDRH3: GGYWYATSYYFDY E3 light chain extended CDRs (SEQ ID NO: 6)CDRL1: RASQSISNNLN (SEQ ID NO: 7) CDRL2: YTSRFHS (SEQ ID NO: 8)CDRL3: QQEHTLPYT Mouse monoclonal antibody 911 extended CDRs911 heavy chain extended CDRs (SEQ ID NO: 9) CDRH1: GFSLIGYDIN(SEQ ID NO: 10) CDRH2: MIWGDGTTDYNSALKS (SEQ ID NO: 11)CDRH3: GGYYYGTSYYFDY 911 light chain extended CDRs (SEQ ID NO: 12)CDRL1: RASQDISNHLN (SEQ ID NO: 13) CDRL2: YISRFHS (SEQ ID NO: 14)CDRL3: QQSKTLPYT E3 heavy chain amino acid sequence (full)(SEQ ID NO: 16) QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK3E light chain amino acid sequence (full antibody) (SEQ ID NO: 17)DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECOther additional CDR sequences referred to (SEQ ID NO: 18) RASQSISNNLN(SEQ ID NO: 19) YTSRFHS (SEQ ID NO: 30) GFSLIGYDLN (SEQ ID NO: 31)IIWGDGTTDYNSAV (SEQ ID NO: 55) QQEHTLPYT (SEQ ID NO: 56) GGYWYATSYYFDY(SEQ ID NO: 57) QQESTLPYT (SEQ ID NO: 58) GGYWYSTSYYFDY (SEQ ID NO: 59)QQEKTLPYT (SEQ ID NO: 60) GGYYYATSYYFDY (SEQ ID NO: 61) QQERTLPYT(SEQ ID NO: 62) GGYWYATSYYFDY (SEQ ID NO: 63) QQERTLPYT (SEQ ID NO: 64)GGYYYATSYYFDY 3E heavy chain nucleotide sequence (full antibody)(SEQ ID NO: 65) CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCCGAGACCCTGTCCCTCACCTGCACTGTCTCTGGGTTCTCACTTATCGGCTATGATCTTAACTGGATCCGACAGCCTCCAGGGAAGGGACTGGAGTGGATTGGGATTATCTGGGGTGATGGAACCACAGACTATAATTCAGCTGTCAAATCCCGCGTCACCATCTCAAAAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGAGGTTATTGGTACGCCACTAGCTACTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCTGTCTTCCCACTGGCCCCATGCTCCCGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCTACTCCCTCAGCAGCGTGGTGACCGTGCCATCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCAAGCAACACCAAGGTCGACAAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCAGTGGCCGGACCATCCGTGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACTGGTATGTGGACGGAGTGGAGGTGCACAACGCCAAGACCAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAGAGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAAGACCAAGGGACAGCCAAGAGAGCCACAGGTGTATACCCTGCCACCATCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGATTCTATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAACTATAAGACCACCCCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTGTATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAAACGTGTTCTCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGAGCCTGTCCCTGTCTCCAGGAAAGTAA3E heavy chain variable domain nucleotide sequence (SEQ ID NO: 66)CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCCGAGACCCTGTCCCTCACCTGCACTGTCTCTGGGTTCTCACTTATCGGCTATGATCTTAACTGGATCCGACAGCCTCCAGGGAAGGGACTGGAGTGGATTGGGATTATCTGGGGTGATGGAACCACAGACTATAATTCAGCTGTCAAATCCCGCGTCACCATCTCAAAAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGAGGTTATTGGTACGCCACTAGCTACTACTTTGACTACTGGGGCCAGGGCACCCTGGT CACCGTCTCCTCA3E light chain nucleotide sequence (full antibody) (SEQ ID NO: 67)GATATCCAGATGACACAGTCCCCATCCTCCCTGTCTGCCTCTGTGGGTGACCGCGTCACCATCACCTGCCGCGCATCTCAGTCCATTAGCAATAATCTGAACTGGTATCAGCAGAAGCCAGGCAAAGCCCCAAAACTCCTGATCTACTACACCTCACGCTTCCACTCAGGTGTCCCATCACGCTTCAGTGGCAGTGGCTCTGGTACAGATTTCACCTTCACCATTAGCAGCCTGCAACCAGAAGATATTGCCACTTATTACTGCCAACAGGAGCATACCCTTCCATATACCTTCGGTCAAGGCACCAAGCTGGAGATCAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTTCCTCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCACGCGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCAGACTACGAGAAACACMAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGAGTGCTAA3E light chain variable domain nucleotide sequence (SEQ ID NO: 68)GATATCCAGATGACACAGTCCCCATCCTCCCTGTCTGCCTCTGTGGGTGACCGCGTCACCATCACCTGCCGCGCATCTCAGTCCATTAGCAATAATCTGAACTGGTATCAGCAGAAGCCAGGCAAAGCCCCAAAACTCCTGATCTACTACACCTCACGCTTCCACTCAGGTGTCCCATCACGCTTCAGTGGCAGTGGCTCTGGTACAGATTTCACCTTCACCATTAGCAGCCTGCAACCAGAAGATATTGCCACTTATTACTGCCAACAGGAGCATACCCTTCCATATACCTTCGGTCAAGGCACCAAGCTGGAGATCAAACGC

The above sequences and other sequences described herein are provided inthe attached sequence listing.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

1. A liquid composition comprising; at least one antibody, at least onetonicity agent, at least one buffer, at least one chelating agent, atleast one surfactant, wherein the pH of said composition is from 5.8 to6.8.
 2. The liquid composition according to claim 1 consistingessentially of; at least one antibody, at least one tonicity agent, atleast one buffer, at least one chelating agent, at least one surfactant,wherein the pH of said composition is from 5.8 to 6.8.
 3. The liquidcomposition according to claim 1, wherein the tonicity agent is a nonreducing sugar.
 4. The liquid composition according to claim 3, whereinthe reducing sugar is trehalose dihydrate at a concentration of about 1mg/ml to about 100 mg/ml.
 5. The liquid composition according to claim 1wherein the surfactant is a polysorbate, preferably polysorbate
 20. 6.The liquid composition according to claim 5 wherein the concentration ofpolysorbate is from about 0.01 to about 0.15 mg/ml.
 7. The liquidcomposition according to claim 1 wherein the buffer is histidine buffer.8. The liquid composition according to claim 7 wherein the concentrationof histidine buffer is from about 1.0 to about 15 mM.
 9. The liquidcomposition according to claim 1 wherein the chelating agent is disodiumEDTA, preferably of a concentration from about 0.01 to about 0.1 mg/ml.10. The liquid composition according claim 1 wherein the antibodyconcentration is less than or equal to about 50 mg/ml.
 11. The liquidcomposition according to claim 10 wherein the antibody concentration isselected from about 2 mg/ml, about 2.5 mg/ml, about 5 mg/ml, about 10mg/ml, about 20 mg/ml, about 22 mg/ml and about 50 mg/ml.
 12. The liquidcomposition according to claim 1 which further comprises an antioxidantagent and/or a preservative agent.
 13. The liquid composition accordingto claim 1 which does not comprise an antioxidant agent, a preservativeagent or both.
 14. The liquid composition according to claim 1comprising; about 0.5 mg/ml to about 50 mg/ml of at least one antibody,about 1.0 mM to about 15 mM histidine buffer, about 1 mg/ml to about 100mg/ml trehalose dihydrate, about 0.01 to about 0.15 mg/ml PS20, about0.01 to about 0.1 mg/ml. disodium EDTA, wherein said composition is of apH from 5.8 to 6.8.
 15. The liquid composition according to claim 13consisting of; about 0.5 mg/ml to about 50 mg/ml of at least oneantibody, about 1.0 mM to about 15 mM histidine buffer, about 1 mg/ml toabout 100 mg/ml trehalose dihydrate, about 0.01 to about 0.15 mg/mlPS20, about 0.01 to about 0.1 mg/ml. disodium EDTA, wherein saidcomposition is of a pH from 5.8 to 6.8.
 16. The liquid compositionaccording to claim 14 comprising; about 2.5 mg/ml, 5 mg/ml, 10 mg/ml, 20mg/ml, 22 mg/ml or about 50 mg/ml of at least one antibody, about 10 mMhistidine buffer, about 84 mg/ml trehalose dihydrate, about 0.1 mg/mlPS20, about 0.05 mg/ml. disodium EDTA, wherein said composition is of apH 6.0+/−0.2.
 17. The liquid composition according to claim 14comprising; about 2.5 mg/ml, 5 mg/ml, 10 mg/ml, 20 mg/ml, 22 mg/ml orabout 50 mg/ml of at least one antibody, about 10 mM histidine buffer,about 84 mg/ml trehalose dihydrate, about 0.1 mg/ml PS20, about 0.05mg/ml disodium EDTA, wherein said composition is of a pH from 5.8 to6.5.
 18. The liquid composition according to claim 1 wherein the atleast one antibody is a human or humanised monoclonal antibody.
 19. Theliquid composition according to claim 1 wherein the at least oneantibody is an IgG2 antibody
 20. The liquid composition according toclaim 1 wherein at least one antibody binds to human NGF with a Kd ofless than or about 2 nM.
 21. The liquid composition according to claim 1wherein the at least one antibody comprises an amino acid sequence thatis at least 90% identical to a heavy chain variable region amino acidsequence shown in SEQ ID NO: 1, and an amino acid sequence that is atleast 90% identical to a light chain variable region amino acid sequenceshown in SEQ ID NO: 2
 22. The liquid composition according to claim 1wherein the at least one antibody comprises an amino acid sequence thatis at least 90% identical to a heavy chain amino acid sequence shown inSEQ ID NO: 16, and an amino acid sequence that is at least 90% identicalto a light chain amino acid sequence shown in SEQ ID NO:
 17. 23. Theliquid composition according to claim 1 wherein the antibody comprises avariable heavy chain sequence of SEQ ID NO. 1 and a variable light chainsequence of SEQ ID NO.
 2. 24. The liquid composition according to claim1 wherein the composition is not lyophilized.
 25. The liquid compositionaccording to claim 1 wherein the composition is resistant to aggregationof the antibody after multiple freeze thaw cycles.
 26. The liquidcomposition according to claim 1 wherein the composition can be storedfor a period of at least about 26 weeks at a temperature of about 40° C.and wherein there is less than about 10% increase in aggregation of theantibody of the composition.
 27. The liquid composition according toclaim 1 wherein the composition can be stored for a period of at leastabout 26 weeks at a temperature of about 40° C. and wherein there isless than about 10% increase in oxidation of the antibody of thecomposition.
 28. The liquid composition according to claim 1 wherein thecomposition can be stored for a period of at least about 26 weeks at atemperature of about 40° C. and wherein there is less than about 10%decrease in activity of the antibody of the composition.
 29. The liquidcomposition according to claim 1 wherein the composition can stored fora period of at least about 26 weeks at a temperature of between 2 to 8°C. and wherein there is less than about 10% increase in aggregation ofthe antibody of the composition.
 30. A pharmaceutical composition fortreating pain in a mammal comprising an effective amount of the liquidcomposition of claim
 1. 31. A method for treating pain in a mammalcomprising administering an effective amount of the liquid compositionof claim 1 to said mammal once every eight weeks.
 32. The methodaccording to claim 31 wherein the volume of the effective amount of theliquid composition of claim 1 is less than or equal to about 2.5 ml. 33.The method according to claim 31 wherein the effective amount of theliquid composition of claim 1 contains less than or equal to 50 mg ofantibody.
 34. The method according to claim 31 wherein administration ofthe effective amount of the liquid composition of claim 1 isadministered either intravenously or subcutaneously intravenously.